Pyrrolobenzodiazepine prodrugs and antibody conjugates thereof

ABSTRACT

The invention relates generally to pyrrolobenzodiazepine monomer and dimer prodrugs having a glutathione-activated disulfide prodrug moiety, a DT-diaphorase-activated quinone prodrug moiety or a reactive oxygen species-activated aryl boronic acid or aryl boronic ester prodrug moiety. The invention further relates to pyrrolobenzodiazepine prodrug dimer-antibody conjugates.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of International Application No.PCT/US2017/046102 filed Aug. 9, 2017, and claims priority benefit ofU.S. Provisional Application Ser. No. 62/373,740 filed on Aug. 11, 2016,each of which are incorporated herein in their entirety.

FIELD OF THE DISCLOSURE

The field of the disclosure relates generally to pyrrolobenzodiazepineprodrugs having a disulfide trigger, a cyclic dione trigger, or an arylboronic acid or aryl boronic ester trigger, and antibody conjugatesthereof.

BACKGROUND

Pyrrolobenzodiazepines (PBD) and dimers thereof are known to interactwith DNA and are effective cancer chemotherapy agents. Problematically,side effects associated with some PBDs, such as cardiotoxicity and acutetissue necrosis have limited use, dosage and effectiveness.

Conjugates comprising a selective carrier-linker-PBD structure (e.g., anantibody-PBD conjugate (ADC)), are attractive selectivechemo-therapeutic molecules, as they combine ideal properties ofselectivity to a target cell and cytotoxic drugs. By directing potentcytotoxic drugs to a target cell, the desired therapeutic effect may beimproved in the target cell while minimizing the effect on non-targetedcells. Examples of such improvements include reduced dose required toachieve a therapeutic effect, targeted delivery, and improvedbloodstream stability. Nonetheless, PBD ADCs may still present adverseside effects that limit use and/or dosage.

A need therefore exists for PBD compounds and formulations that providedfor reduced toxicity and improved bioefficacy.

SUMMARY

In some embodiments, a PBD prodrug dimer-antibody conjugate compositionof formula (I) comprising a first PBD prodrug monomer M1 and a secondPBD-antibody monomer M2 is provided:

M1 is a PBD monomer. R² is selected from —H, ═CH₂, —CN, —R, ═CH—R, aryl,heteroaryl, bicyclic ring and heterobicyclic ring. R³ is H. R⁶, R⁷ andR⁹ are independently selected from H, R, OH, OR, halo, amino, nitro, SHand SR. X is selected from S, O and NH. R¹⁰ is a prodrug moietycomprising (i) a glutathione-activated disulfide, (ii) aDT-diaphorase-activated quinone, or (iii) a reactive oxygenspecies-activated aryl boronic acid or aryl boronic ester. R¹¹ isselected from (i) H and R when X is O or NH, and (ii) H, R and O_(z)Uwhen X is S, wherein z is 2 or 3 and U is a monovalent pharmaceuticallyacceptable cation. R is selected from a lower alkyl group having 1 to 10carbon atoms and an arylalkyl group of up to 12 carbon atoms, (i)wherein the alkyl group optionally contains one or more carbon-carbondouble or triple bonds, or an arylalkyl, of up to 12 carbon atoms and(ii) wherein R is optionally substituted by one or more halo, hydroxy,amino, or nitro groups, and optionally contains one or more heteroatoms. M1 contains an optional double bond, as indicated by the dashedline, between one of: (i) C₁ and C₂; (ii) C₂ and C₃; and (iii) C₂ andR².

M2 is a PBD monomer. R^(2′), R^(3′), R^(6′), R^(7′), R^(9′), R^(11′) andX′ correspond to, and are defined in the same way as, R², R³, R⁶, R⁷,R⁹, R¹¹ and X, respectively. L is a self-immolative linker comprising adisulfide moiety, a peptide moiety or a peptidomimetic moiety. M2contains an optional double bond, as indicated by the dashed line,between one of: (i) C_(1′) and C_(2′); (ii) C_(2′) and C_(3′); and (iii)C_(2′) and R^(2′).

M1 and M2 are bound at the C8 position by a moiety -Q-T-Q′-, wherein Qand Q′ are independently selected from O, NH and S, and wherein T is anoptionally substituted C₁₋₁₂ alkylene group that is further optionallyinterrupted by one or more heteroatoms and/or aromatic rings. Ab is anantibody, and p is an integer having a value of 1, 2, 3, 4, 5, 6, 7 or8, and represents the number of PBD prodrug dimers that may beconjugated or bound to the antibody. Each asterisk independentlyrepresents a chiral center of racemic or undefined stereochemistry.

In some other embodiments, a PBD monomer prodrug composition of formula(II) is provided.

R² is selected from —H, ═CH₂, —CN, —R, ═CH—R, aryl, heteroaryl, bicyclicring and heterobicyclic ring. R³ is H. R⁶, R⁷, R⁸ and R⁹ areindependently selected from H, R, OH, OR, halo, amino, nitro, SH and SR,or R⁷ and R⁸ together with the carbon atoms to which they are bound forma group —O—(CH₂)_(n)—O—, where n is 1 or 2. X is selected from S, O andNH. R¹¹ is a prodrug moiety comprising a (i) glutathione-activateddisulfide, (ii) a DT-diaphorase-activated quinone or (iii) a reactiveoxygen species-activated aryl boronic acid or aryl boronic ester. R¹¹ isselected from (i) H and R when X is O or NH, and (ii) H, R and O_(z)Uwhen X is S, wherein z is 2 or 3 and U is a monovalent pharmaceuticallyacceptable cation. R is selected from a lower alkyl group having 1 to 10carbon atoms and an arylalkyl group of up to 12 carbon atoms, (i)wherein the alkyl group optionally contains one or more carbon-carbondouble or triple bonds, or an aryl group, of up to 12 carbon atoms and(ii) wherein R is optionally substituted by one or more halo, hydroxy,amino, or nitro groups, and optionally contains one or more heteroatoms. The PBD monomer prodrug contains an optional double bond, asindicated by the dashed line, between one of: (i) C₁ and C₂; (ii) C₂ andC₃; and (iii) C₂ and R². Each asterisk independently represents a chiralcenter of racemic or undefined stereochemistry.

In some other embodiments, a PBD prodrug dimer compound of formula(VIII) comprising a first PBD prodrug monomer M1 and a second PBDmonomer M2 is provided:

M1 is a PBD monomer. R² is selected from —H, ═CH₂, —CN, —R, ═CH—R, aryl,heteroaryl, bicyclic ring and heterobicyclic ring. R³ is H. R⁶, R⁷ andR⁹ are independently selected from H, R, OH, OR, halo, amino, nitro, SHand SR. X is selected from S, O and NH. R¹⁰ is a prodrug moietycomprising (i) a glutathione-activated disulfide, (ii) aDT-diaphorase-activated quinone or (iii) a reactive oxygenspecies-activated aryl boronic acid or aryl boronic ester. R¹¹ isselected from (i) H and R when X is O or NH, and (ii) H, R and O_(z)Uwhen X is S, wherein z is 2 or 3 and U is a monovalent pharmaceuticallyacceptable cation. R is selected from a lower alkyl group having 1 to 10carbon atoms and an arylalkyl group of up to 12 carbon atoms, (i)wherein the alkyl group optionally contains one or more carbon-carbondouble or triple bonds, or an aryl group, of up to 12 carbon atoms and(ii) wherein R is optionally substituted by one or more halo, hydroxy,amino, or nitro groups, and optionally contains one or more heteroatoms. M1 contains an optional double bond, as indicated by the dashedline, between one of: (i) C₁ and C₂; (ii) C₂ and C₃; and (iii) C₂ andR².

M2 is a PBD monomer. R^(2′), R^(3′), R^(6′), R^(7′), R^(9′), R^(11′) andX′ correspond to R², R³, R⁶, R⁷, R⁹, R¹¹ and X, respectively. M2contains an optional double bond, as indicated by the dashed line,between one of: (i) C_(1′) and C_(2′); (ii) C_(2′) and C_(3′); and (iii)C_(2′) and R^(2′). R¹² is absent when the bond between N10′ and C11′ isa double bond, or is selected from —C(O)O-L and —C(O)O—R¹⁰.

L is a self-immolative linker comprising at least one of a disulfidemoiety, a peptide moiety and a peptidomimetic moiety. M1 and M2 arebound at the C8 position by a moiety -Q-T-Q′-, wherein Q and Q′ areindependently selected from O, NH and S, and wherein T is an optionallysubstituted C₁₋₁₂ alkylene group that is further optionally interruptedby one or more heteroatoms and/or aromatic rings.

Each asterisk independently represents a chiral center of racemic orundefined stereochemistry.

In some other embodiments, a pharmaceutical composition comprising thePBD prodrug dimer-antibody conjugate compound previously described and apharmaceutically acceptable diluent, antibody or excipient is provided.

In some other embodiments, a method of treating cancer comprisingadministering to a patient a pharmaceutical composition as previouslydescribed is provided.

In other embodiments, a use of an antibody-drug conjugate compound aspreviously described in the manufacture of a medicament for thetreatment of cancer in a mammal is provided.

In still other embodiments, an antibody-drug conjugate compound aspreviously described for use in a method for treating cancer isprovided.

In other embodiments, an article of manufacture comprising apharmaceutical composition as previously described, a container, and apackage insert or label indicating that the pharmaceutical compositioncan be used to treat cancer is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a plot of PBD monomer disulfide prodrug Activity [%]against KPL-4 cells versus the log of prodrug concentration in moles perliter and further depicts a table of PBD monomer IC₅₀ potency againstthe KPL-4 cells.

FIG. 2 depicts a plot of PBD monomer disulfide prodrug Activity [%]against WSU-DLCL2 cells versus the log of prodrug concentration in molesper liter and further depicts a table of PBD monomer IC₅₀ potencyagainst the WSU-DLCL2 cells.

FIG. 3 depicts a plot of SK-BR-3 cell viability (% of control) versusPBD monomer disulfide prodrug concentration on a g/mL basis.

FIG. 4 depicts a plot of PBD monomer disulfide prodrug stability inhuman and rat whole blood evaluated at 4- and 24-hour intervals wherethe results are presented as percent of the parent compound remainingrelative to time zero.

FIG. 5 depicts a plot of SK-BR-3 cell viability (% of control) versusPBD monomer disulfide prodrug concentration in micromoles and PBD dimerdisulfide prodrug concentration in μg/mL.

FIG. 6 depicts a plot of KPL-4 cell viability (% of control) versus PBDmonomer disulfide prodrug concentration in micromoles and PBD dimerdisulfide prodrug concentration in μg/mL.

FIG. 7 depicts a plot of UACC-257 and IGROV-1 relative cell viability (%of control) versus a PBD dimer non-prodrug control concentration in nMand further depicts a table of PBD dimer IC₅₀ potency against theUACC-257 and IGROV-1 cells.

FIG. 8 depicts a plot of UACC-257 and IGROV-1 relative cell viability (%of control) versus the concentration in nM of a PBD dimer having adisulfide prodrug a one PBD monomer N10 position and not having aprodrug at the other PBD monomer and further depicts a table of PBDdimer IC₅₀ potency and IC₅₀ ratio for an indicated GSH concentrationagainst the UACC-257 and IGROV-1 cells. The IC₅₀ ratio is determinedrelative to the data for the PBD dimer non-prodrug control depicted inFIG. 7.

FIG. 9 depicts a plot of UACC-257 and IGROV-1 relative cell viability (%of control) versus the concentration in nM of a PBD dimer having adisulfide prodrug at the N10 position of both PBD monomers and furtherdepicts a table of PBD dimer IC₅₀ potency and IC₅₀ ratio for anindicated GSH concentration against the UACC-257 and IGROV-1. The IC₅₀ratio is determined relative to the data for the PBD dimer non-prodrugcontrol depicted in FIG. 7.

FIG. 10 depicts a plot of SK—BR3 relative cell viability (% of control)versus the concentration in nM of (i) PBD dimer non-prodrug, (ii) a PBDdimer having a disulfide prodrug a one PBD monomer N10 position and nothaving a prodrug at the other PBD monomer, and (iii) a PBD dimer havinga disulfide prodrug at the N10 position of both PBD monomers.

FIG. 11 depicts a plot of SK-BR-3 cell viability (% of control) versusthe concentration of (i) a 7C2 HC A140C peptide-linked disulfidecyclopentyl prodrug ADC PBD dimer, (ii) a 7C2 LC K149C peptide-linkeddisulfide thio-phenol prodrug ADC PBD dimer, (iii) a 7C2 HC A140C ADCPBD dimer not having a prodrug moiety, and (iv) a CD22 HC A140C ADC PBDdimer not having a prodrug moiety.

FIG. 12 depicts a plot of KPL-4 cell viability (% of control) versus theconcentration of (i) a 7C2 HC A140C peptide-linked disulfide cyclopentylprodrug ADC PBD dimer, (ii) a 7C2 LC K149C peptide-linked disulfidethio-phenol prodrug ADC PBD dimer, (iii) a 7C2 HC A140C ADC PBD dimernot having a prodrug moiety, and (iv) a CD22 HC A140C ADC PBD dimer nothaving a prodrug moiety.

FIG. 13 depicts a plot of SK-BR-3 cell viability (% of control) versusthe concentration of (i) 7C2 LC K149C peptide linked disulfidecyclobutyl prodrug ADC PBD dimer, (ii) 7C2 LC K149C peptide linkeddisulfide cyclopentyl prodrug ADC PBD dimer, (iii) 7C2 LC K149C peptidelinked disulfide thio-phenol prodrug ADC PBD dimer, (iv) 7C2 LC K149Cpeptide linked disulfide isopropyl prodrug ADC PBD dimer, (v) 4D5 HCA118C ADC PBD dimer not having a prodrug moiety, and (vi) 4D5 LC V205CADC PBD dimer not having a prodrug moiety.

FIG. 14 depicts a plot of KPL-4 cell viability (% of control) versus theconcentration of (i) 7C2 LC K149C peptide linked disulfide cyclobutylprodrug ADC PBD dimer, (ii) 7C2 LC K149C peptide linked disulfidecyclopentyl prodrug ADC PBD dimer, (iii) 7C2 LC K149C peptide linkeddisulfide thio-phenol prodrug ADC PBD dimer, (iv) 7C2 LC K149C peptidelinked disulfide isopropyl prodrug ADC PBD dimer, (v) 4D5 HC A118C ADCPBD dimer not having a prodrug moiety, and (vi) 4D5 LC V205C ADC PBDdimer not having a prodrug moiety.

FIG. 15A depicts a plot of WSU-DLCL normalized percent of viable cellsafter 3 days as compared to the number of cells at time zero versus theconcentration of (i) a CD22 antibody ADC PBD dimer having a benzylformate (C₆H₅—CH₂—O—C(O)—) moiety at the N10 position of one PBD monomer(negative control), (ii) a CD22 antibody ADC PBD dimer aryl boronic acidprodrug ((OH)₂B—C₆H₄—CH₂—O—C(O)—), and (iii) a CD22 antibody ADC PBDdimer not having a prodrug moiety (positive control).

FIG. 15B depicts a plot of WSU-DLCL cell kill versus drug concentrationin μg/mL three days after exposure to: (i) a CD22 antibody ADC PBD dimerhaving a boronic acid prodrug (PBD dimer ADC boronic acid prodrug 1A);(ii) a Ly6E antibody ADC PBD dimer having a boronic acid prodrug (PBDdimer ADC boronic acid prodrug 1B); (iii) a CD22 antibody ADC PBD dimernot having a prodrug moiety (PBD dimer ADC boronic acid control 2)(positive control) and (iv) a blank control.

FIG. 15C depicts a plot of MDA-MB-453 cell kill versus drugconcentration in μM three days after exposure to: (i) a PBD monomercontrol; (ii) a PBD monomer control having a benzyl formate moiety atthe N10 PBD position; and (iii) a PBD monomer boronic acid prodrug.

FIG. 15D depicts a plot of MDA-MB-453 cell kill versus drugconcentration in μM three days after exposure to: (i) silvestrol, (ii) aPBD monomer control; (iii) a PBD monomer control having a benzyl formatemoiety at the N10 PBD position; (iv) a PBD monomer boronic acid prodrug;(v) the PBD monomer control and silvestrol; (vi) the PBD monomer controlhaving a benzyl formate moiety at the N10 PBD position and silvestrol;and (vii) the PBD monomer boronic acid prodrug and silvestrol.7

FIG. 16 depicts a plot of BJAB normalized percent of viable cells after3 days as compared to the number of cells at time zero versus theconcentration of (i) a CD22 antibody ADC PBD dimer having a benzylformate (C₆H₅—CH₂—O—C(O)—) moiety at the N10 position of one PBD monomer(negative control), (ii) a CD22 antibody ADC PBD dimer aryl boronic acidprodrug ((OH)₂B—C₆H₄—CH₂—O—C(O)—), and (iii) a CD22 antibody ADC PBDdimer not having a prodrug moiety (positive control).

FIG. 17 depicts a plot of the Activity [%] against KPL-4 cells versusthe concentration in M of (i) a PBD dimer having a quinone prodrug atthe N10 position of one PBD monomer and no prodrug or linker at the N10position of the other PBD monomer and (ii) a PBD dimer not having aprodrug or a linker. FIG. 17 further depicts a table of PBD dimerdiaphorase prodrug and control IC₅₀ potency and IC₅₀ ratio against theKPL-4 cells. The IC₅₀ ratio is based on the prodrug IC₅₀ value relativeto the PBD dimer control.

FIG. 18 depicts a plot of the Activity [%] against WSU cells versus theconcentration in M of (i) a PBD dimer having a quinone prodrug at theN10 position of one PBD monomer and no prodrug or linker at the N10position of the other PBD monomer and (ii) a PBD dimer not having aprodrug or a linker. FIG. 18 further depicts a table of PBD dimerdiaphorase prodrug and control IC₅₀ potency and IC₅₀ ratio against theKPL-4 cells. The IC₅₀ ratio is based on the prodrug IC₅₀ value relativeto the PBD dimer control.

FIG. 19 depicts a plot of the Activity [%] against KPL-4 cells versusthe concentration in M of (i) a PBD monomer having a quinone prodrug atthe N10 and (ii) a PBD monomer not having a prodrug or a linker. FIG. 19further depicts a table of PBD dimer diaphorase prodrug and control IC₅₀potency and IC₅₀ ratio against the KPL-4 cells. The IC₅₀ ratio is basedon the prodrug IC₅₀ value relative to the PBD dimer control.

FIG. 20 depicts a plot of the Activity [%] against WSU cells versus theconcentration in M of (i) a PBD monomer having a quinone prodrug at theN10 and (ii) a PBD monomer not having a prodrug or a linker. FIG. 20further depicts a table of PBD dimer diaphorase prodrug and control IC₅₀potency and IC₅₀ ratio against the WSU cells. The IC₅₀ ratio is based onthe prodrug IC₅₀ value relative to the PBD dimer control.

FIG. 21 depicts a plot of SK-BR-3 cell viability (% of control) versusthe concentration in μg/mL of (i) 7C2 LC K149C VC-PBD DT diaphorasequinone prodrug ADC PBD dimer, (ii) Ly6E LC K149C VC-PBD DT diaphorasequinone prodrug ADC PBD dimer, and (iii) 4D5 HC A118C ADC PBD dimer nothaving a prodrug moiety.

FIG. 22 depicts a plot of KPL-4 cell viability (% of control) versus theconcentration in μg/mL of (i) 7C2 LC K149C VC-PBD DT diaphorase quinoneprodrug ADC PBD dimer, (ii) Ly6E LC K149C VC-PBD DT diaphorase quinoneprodrug ADC PBD dimer, and (iii) 4D5 HC A118C ADC PBD dimer not having aprodrug moiety.

FIG. 23 depicts a plot of tumor volume (mm³) versus days after treatmentfor SCID mice with BJAB-luc human Burkitt's lymphoma with: (i) histidinebuffer vehicle; (ii) non-prodrug anti-CD22 HC-A118C PBD dimer ADC; (iii)anti-CD22 LC-K149C PDB dimer boronic acid prodrug ADC; (iv) anti-Ly6ELC-K149C PDB dimer boronic acid prodrug ADC; (v) non-prodrug anti-CD22LC-K149C PDB dimer ADC; and (vi) non-prodrug anti-Her2 HC-A118C PBDdimer ADC.

FIG. 24 depicts a plot of % body weight change versus days aftertreatment for SCID mice with BJAB-luc human Burkitt's lymphoma with: (i)histidine buffer vehicle; (ii) non-prodrug anti-CD22 HC-A118C PBD dimerADC; (iii) anti-CD22 LC-K149C PDB dimer boronic acid prodrug ADC; (iv)anti-Ly6E LC-K149C PDB dimer boronic acid prodrug ADC; (v) non-prodruganti-CD22 LC-K149C PDB dimer ADC; and (vi) non-prodrug anti-Her2HC-A118C PBD dimer ADC.

FIG. 25 depicts a plot of tumor volume (mm³) versus days after treatmentfor SCID-beige mice with KPL-4 human breast tumors with: (i) vehicle;(ii) non-prodrug anti-Her2 HC-A140C PBD dimer ADC; and (iii) anti-Her2HC-A140C PBD thio-phenol prodrug ADC.

FIG. 26 depicts a plot of % body weight change versus days aftertreatment for SCID-beige mice with KPL-4 human breast tumors with: (i)vehicle; (ii) non-prodrug anti-Her2 HC-A140C PBD dimer ADC; and (iii)anti-Her2 HC-A140C PBD thio-phenol prodrug ADC.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

In some embodiments, the present disclosure is generally directed to PBDmonomer prodrug compounds of formula (II):

where R², R³, R⁶, R⁷, R⁸, R⁹, X, R¹⁰, R¹¹ and * are defined in moredetail elsewhere herein.

In some embodiments, the present disclosure is directed to PBD dimerprodrug compounds comprising a first PBD monomer having R¹⁰ at the N10position. The dimer additionally comprises a second PBD monomer havingat the N10 position: (1) no substitution; (2) R¹⁰; or (3) a linker. ThePBD dimer is generally one of the following two structures:

where R², R^(2′), R³, R^(3′), R⁶, R^(6′), R⁷, R^(7′), R⁹, R^(9′), X,R¹⁰, R¹¹, R^(11′), Q, Q′, T, *, and the linker are defined in moredetail elsewhere herein.

In some embodiments, the present disclosure is directed to PBD dimerprodrug compounds comprising a first PBD monomer having at the N10position: (i) a protecting group comprising a GSH-activated disulfidetrigger, (ii) a protecting group comprising a DTD-activated quinonetrigger, or (iii) a protecting group comprising a ROS-activated arylboronic acid or aryl boronic ester trigger. The dimer additionallycomprises a second PBD monomer having a linker conjugated to an antibodysulfhydryl moiety at the N10 position. The PBD dimer is as generallyfollows:

where R², R^(2′), R³, R^(3′), R⁶, R^(6′), R⁷, R^(7′), R⁹, R^(9′), X,R¹⁰, R¹¹, R^(11′), T, *, the linker, the antibody, and p are defined inmore detail elsewhere herein.

I. Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs, and are consistent with:Singleton et al. (1994) Dictionary of Microbiology and MolecularBiology, 2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C.,Travers, P., Walport, M., Shlomchik (2001) Immunobiology, 5th Ed.,Garland Publishing, New York.

A “prodrug” as defined herein is a PBD substituted at the N10 positionwith a protecting group comprising a trigger, wherein the protectinggroup masks drug toxicity. The protecting group is enzymatically orchemically activated (cleaved) to generate the active drug by theapplication of stimulus to the trigger, such as an enzyme (e.g., DTD),ROS or GSH. In some embodiments the trigger is a disulfide, a cyclicdione (e.g., a quinone), or an aryl boronic acid or an aryl boronicester.

A “protecting group” as defined herein refers to a moiety introducedinto a drug molecule by chemical modification of a functional group thatblocks or protects a particular functionality.

“DTD” refers to DT-diaphorase; “ROS” refers to a reactive oxygenspecies; and “GSH” refers to glutathione.

A “linker” (L) is a bifunctional or multifunctional moiety that can beused to link one or more drug moieties (D) to an antibody (Ab) to forman antibody-linker-drug conjugate of the general formula:Antibody-[L-D]_(p)wherein p may be 1, 2, 3, 4, 5, 6, 7 or 8. The linker generallycomprises a connection to the antibody (Ab), an optional antibody spacerunit, an optional trigger unit to provide for immolation, an optionaldrug (D) spacer unit, and a connection to the drug, and is of thegeneral structure:Ab-[Ab connection]-[Ab spacer]_(opt)-[Trigger]_(opt)-[D spacer]_(opt)-[Dconnection]-D.

In some embodiments, antibody-D conjugates can be prepared using alinker having reactive functionalities for covalently attaching to thedrug and to the antibody. For example, in some embodiments, the cysteinethiol of a cysteine-engineered antibody (Ab) can form a bond with areactive functional group of a linker or a drug-linker intermediate tomake an ADC. In one embodiment, a linker has a functionality that iscapable of reacting with a free cysteine present on an antibody to forma covalent disulfide bond (See, e.g., the conjugation method at page 766of Klussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, and theExamples herein, incorporated herein by reference in its entirety). Insome embodiments, the linker may comprise a cleavable immolative moietysuch as a peptide, peptidomimetic or disulfide trigger. A linker mayoptionally comprise one or more “spacer” units between an immolativemoiety and the drug moiety (such as p-amino-benzyl (“PAB”)) and/orbetween an immolative moiety and the antibody (such as a moiety derivedfrom caproic acid). Non-limiting examples of spacers includevaline-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe”), and p-aminobenzyloxycarbonyl (a “PABC”). In someembodiments the spacer may be immolating.

“Immolating” and “immolative” refer to a moiety, such as a linker,spacer and/or prodrug trigger, that is cleavable in vivo and/or in vitrosuch as by an enzyme (e.g. a protease or DTD), GSH, a ROS, and/or pHchange. Examples of immolative moieties include disulfides, peptides,and peptidomimetics.

“Peptide” refers to short chains of two or more amino acid monomerslinked by amide (peptide) bonds. The amino acid monomers may benaturally occurring and/or non-naturally occurring amino acid analogs.

“Peptidomimetic” refers to a group or moiety that has a structure thatis different from the general chemical structure of an amino acid orpeptide, but functions in a manner similar to a naturally occurringamino acid or peptide.

“Hindered linker” refers to a linker having a carbon atom bearing asulfur capable of forming a disulfide bond wherein the carbon atom issubstituted with at least one substituent other than H, and moreparticularly is substituted with a hydrocarbyl or a substitutedhydrocarbyl moiety as further detailed herein below.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein.

“Cell Targeting Moiety” refers to an antibody having binding affinityfor a target expressing an antigen.

“Predominantly Comprises” refers to at least 50%, at least 75%, at least90%, at least 95% or at least 99% of a referenced component on a recitedbasis, such as for instance and without limitation, w/w %, v/v %, w/v %,mole % or equivalent % basis. “Consisting essentially of” generallylimits a feature, compound, composition or method to the recitedelements and/or steps, but does not exclude the possibility ofadditional elements and/or steps that do not materially affect thefunction, compound, composition and/or characteristics of the recitedfeature, compound, composition or method.

The terms “antibody” and “Ab” herein are used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al. (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein generated by theimmune system that is capable of recognizing and binding to a specificantigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). A target antigengenerally has numerous binding sites, also called epitopes, recognizedby CDRs on multiple antibodies. Each antibody that specifically binds toa different epitope has a different structure. Thus, one antigen mayhave more than one corresponding antibody. An antibody includes afull-length immunoglobulin molecule or an immunologically active portionof a full-length immunoglobulin molecule, i.e., a molecule that containsan antigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies. In one embodiment, however, the immunoglobulin is of human,murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)2, and Fv fragments;diabodies; linear antibodies; minibodies (Olafsen et al. (2004) ProteinEng. Design & Sel. 17(4):315-323), fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies, CDR(complementary determining region), and epitope-binding fragments of anydescribed herein which immunospecifically bind to cancer cell antigens,viral antigens or microbial antigens, single-chain antibody molecules;and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present disclosure may be made by the hybridoma method firstdescribed by Kohler et al. (1975) Nature 256:495, or may be made byrecombinant DNA methods (see for example: U.S. Pat. Nos. 4,816,567;5,807,715). The monoclonal antibodies may also be isolated from phageantibody libraries using the techniques described in Clackson et al.(1991) Nature, 352:624-628; Marks et al. (1991) J. Mol. Biol.,222:581-597; for example.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal. (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimericantibodies of interest herein include “primatized” antibodies comprisingvariable domain antigen-binding sequences derived from a non-humanprimate (e.g., Old World Monkey, Ape, etc.) and human constant regionsequences.

An “intact antibody” herein is one comprising a VL and VH domains, aswell as a light chain constant domain (CL) and heavy chain constantdomains, CH1, CH2 and CH3. The constant domains may be native sequenceconstant domains (e.g., human native sequence constant domains) or aminoacid sequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact immunoglobulin antibodies: IgA,IgD, IgE, IgG, and IgM, and several of these may be further divided into“subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.The heavy-chain constant domains that correspond to the differentclasses of antibodies are called α, δ, ε, γ, and μ, respectively. Thesubunit structures and three-dimensional configurations of differentclasses of immunoglobulins are well known. Ig forms includehinge-modifications or hingeless forms (Roux et al. (1998) J. Immunol.161:4083-4090; Lund et al. (2000) Eur. J. Biochem. 267:7246-7256; US2005/0048572; US 2004/0229310).

A “cysteine engineered antibody” or “cysteine engineered antibodyvariant” is an antibody in which one or more residues of an antibody aresubstituted with cysteine residues. In accordance with the presentdisclosure, the thiol group(s) of the cysteine engineered antibodies canbe conjugated to prodrugs of the disclosure to form a THIOMAB™ ADC(i.e., a THIOMAB™ drug conjugate (TDC)). In particular embodiments, thesubstituted residues occur at accessible sites of the antibody. Bysubstituting those residues with cysteine, reactive thiol groups arethereby positioned at accessible sites of the antibody and may be usedto conjugate the antibody to the drug moiety to create animmunoconjugate, as described further herein. For example, a THIOMAB™antibody may be an antibody with a single mutation of a non-cysteinenative residue to a cysteine in the light chain (e.g., G64C, K149C orR142C according to Kabat numbering) or in the heavy chain (e.g., D101Cor V184C or T205C according to Kabat numbering). In specific examples, aTHIOMAB™ antibody has a single cysteine mutation in either the heavy orlight chain such that each full-length antibody (i.e., an antibody withtwo heavy chains and two light chains) has two engineered cysteineresidues. Cysteine engineered antibodies and preparatory methods aredisclosed by US 2012/0121615 A1 (incorporated by reference herein in itsentirety).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include, butare not limited to, carcinoma, lymphoma (e.g., Hodgkin's andnon-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include acute myeloid leukemia(AML), myelodysplastic syndrome (MDS), chronic myelogenous leukemia(CML), chronic myelomonocytic leukemia, acute promyelocytic leukemia(APL), chronic myeloproliferative disorder, thrombocytic leukemia,precursor B-cell acute lymphoblastic leukemia (pre-B-ALL), precursorT-cell acute lymphoblastic leukemia (preT-ALL), multiple myeloma (MM),mast cell disease, mast cell leukemia, mast cell sarcoma, myeloidsarcomas, lymphoid leukemia, and undifferentiated leukemia. In someembodiments, the cancer is myeloid leukemia. In some embodiments, thecancer is acute myeloid leukemia (AML).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “epitope” refers to the particular site on an antigen moleculeto which an antibody binds. In some embodiments, the particular site onan antigen molecule to which an antibody binds is determined by hydroxylradical footprinting.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).)A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.Exemplary hypervariable loops occur at amino acid residues 26-32 (L1),50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3).(Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acidresidues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 ofH2, and 95-102 of H3. (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) With the exception of CDR1in VH, CDRs generally comprise the amino acid residues that form thehypervariable loops. CDRs also comprise “specificity determiningresidues,” or “SDRs,” which are residues that contact antigen. SDRs arecontained within regions of the CDRs called abbreviated-CDRs, or a-CDRs.Exemplary a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, anda-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2, 89-96of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See Almagro andFransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwiseindicated, HVR residues and other residues in the variable domain (e.g.,FR residues) are numbered herein according to Kabat et al., supra.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated antibody” is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The terms “treat” and “treatment” refer to both therapeutic treatmentand prophylactic or preventative measures, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder, such as the development or spread of cancer. For purposes ofthis disclosure, beneficial or desired clinical results include, but arenot limited to, alleviation of symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.Those in need of treatment include those already with the condition ordisorder as well as those prone to have the condition or disorder orthose in which the condition or disorder is to be prevented.

The term “therapeutically effective amount” refers to an amount of adrug effective to treat a disease or disorder in a mammal. In the caseof cancer, the therapeutically effective amount of the drug may reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve to some extent one or more of the symptoms associated with thecancer. To the extent the drug may prevent growth and/or kill existingcancer cells, it may be cytostatic and/or cytotoxic. For cancer therapy,efficacy can, for example, be measured by assessing the time to diseaseprogression (TTP) and/or determining the response rate (RR).

The term “leaving group,” as used herein, refers to a moiety that leavesin the course of a chemical reaction involving the groups as describedherein.

The term “hydrocarbyl” as used herein describes organic compounds orradicals consisting exclusively of the elements carbon and hydrogen.These moieties include, without limitation, alkyl, alkenyl, alkynyl, andaryl moieties. These moieties also include alkyl, alkenyl, alkynyl, andaryl moieties substituted with other aliphatic or cyclic hydrocarbongroups, such as alkaryl, alkenaryl and alkynaryl. Unless otherwiseindicated, these moieties preferably comprise 1 to 20 carbon atoms, 1 to10 carbon atoms or 1 to 6 carbon atoms.

The term “alkyl”, as used herein, by itself or as part of anothersubstituent, means, unless otherwise stated, a straight or branchedchain hydrocarbon radical, having the number of carbon atoms designated(i.e., C₁₋₈ means one to eight carbons). Examples of alkyl groupsinclude methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl,iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and thelike. Unless otherwise indicated, the alkyl groups described herein arepreferably lower alkyl containing from one to ten or one to eight carbonatoms in the principal chain. They may be straight or branched chain orcyclic including, but not limited to, methyl, ethyl, propyl, isopropyl,allyl, benzyl, hexyl and the like. The alkyl moieties may optionallycomprise one or more hetero atoms selected from O, S and N and arereferred to as “heteroalkyl”.

The terms “carbocycle”, “carbocyclyl”, “carbocyclic ring” and“cycloalkyl” refer to a monovalent non-aromatic, saturated or partiallyunsaturated ring having 3 to 12 carbon atoms (C₃₋₁₂) as a monocyclicring or 7 to 12 carbon atoms as a bicyclic ring. Bicyclic carbocycleshaving 7 to 12 atoms can be arranged, e.g., as a bicyclo [4,5], [5,5],[5,6] or [6,6] system, and bicyclic carbocycles having 9 or 10 ringatoms can be arranged as a bicyclo [5,6] or [6,6] system, or as bridgedsystems such as bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane andbicyclo[3.2.2]nonane. Examples of monocyclic carbocycles include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl,cyclohexadienyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl,cycloundecyl, cyclododecyl, and the like. The carbocycle and cycloalkylmoieties may optionally comprise one or more hetero atoms selected fromO, S and N.

The term “alkoxy” refers to those alkyl groups attached to the remainderof the molecule via an oxygen atom. The alkoxy moieties may optionallycomprise one or more hetero atoms selected from O, S and N and arereferred to as “heteroalkoxy”.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, such as —CH₂CH₂CH₂CH₂CH₂—.

Unless otherwise indicated, the alkynyl groups described herein arepreferably lower alkynyl containing from two to eight carbon atoms inthe principal chain and up to 20 carbon atoms. They may be straight orbranched chain including, but not limited to, ethynyl, propynyl,butynyl, isobutynyl, hexynyl, and the like.

The term “aryl” as used herein alone or as part of another group denotesoptionally substituted homocyclic aromatic groups, preferably monocyclicor bicyclic groups containing from 5 to 20 carbons, from 5 to 10carbons, or from 5 to 6 carbons in the ring portion, including, but notlimited to, phenyl, biphenyl, naphthyl, substituted phenyl, substitutedbiphenyl or substituted naphthyl. The aryl moieties may optionallycomprise one or more hetero atoms selected from O, S and N and arereferred to as “heteroaryl” or “heterobicyclic”. Such heteroaromaticsmay comprise 1 or 2 nitrogen atoms, 1 or 2 sulfur atoms, 1 or 2 oxygenatoms, and combinations thereof, in the ring, wherein the each heteroatom is bonded to the remainder of the molecule through a carbon. Nonlimiting exemplary groups include pyridine, pyrazine, pyrimidine,pyrazole, pyrrole, imidazole, thiopene, thiopyrrilium, parathiazine,indole, purine, benzimidazole, quinolone, phenothiazine. Non-limitingexemplary substituents include one or more of the following groups:hydrocarbyl, substituted hydrocarbyl, alkyl, alkoxy, acyl, acyloxy,alkenyl, alkenoxy, aryl, aryloxy, amino, amido, acetal, carbamyl,carbocyclo, cyano, ester, ether, halogen, heterocyclo, hydroxy, keto,ketal, phospho, nitro, and thio.

The term “arylalkyl” as used herein refers to an aryl moiety substitutedwith at least one alkyl, and optionally further substituted. One exampleof arylalkyl is phenylmethyl, also referred to as benzyl (C₆H₅CH₃) orbenzylene (—C₆H₄CH₂—).

The “substituted” moieties described herein are moieties such ashydrocarbyl, alkyl, heteroaryl, bicyclic and heterobicyclic which aresubstituted with at least one atom other than carbon, including moietiesin which a carbon chain atom is substituted with a hetero atom such asnitrogen, oxygen, silicon, phosphorous, boron, sulfur, or a halogenatom. These substituents include, but are not limited to, halogen,heterocyclo, alkoxy, alkenoxy, alkynoxy, aryloxy, hydroxy, keto, acyl,acyloxy, nitro, tertiary amino, amido, nitro, cyano, thio, sulfinate,sulfonamide, ketals, acetals, esters and ethers.

The terms “halogen” and “halo” as used herein alone or as part ofanother group refer to chlorine, bromine, fluorine, and iodine.

The term “cyclic dione” refers to cyclic and heterocyclic compoundshaving an even number of —C(O)— groups. In some embodiments, the cycliccompounds are aryl (quinones). In some embodiments, the heterocycliccompounds heteroaryl. A non-exclusive listing of cyclic diones includesnaphthoquinone and indole dione.

The term “pharmaceutically acceptable cation”, denoted as U, refers to amonovalent cation. Examples of pharmaceutically acceptable monovalentcations are discussed in Berge, et al., J Pharm. Sci., 66, 1-19 (1977),which is incorporated herein by reference. In some aspects, thepharmaceutically acceptable cation is inorganic, including but notlimited to, alkali metal ions (e.g., sodium or potassium ions) andammonia. For instance, in some aspects, the moiety SO_(z)U may be SO₃Na,SO₃K or SO₃NH₄.

Certain compounds of the present disclosure may possess asymmetriccarbon atoms (optical centers) or double bonds. Such compounds have thesame molecular formula but differ in the nature or sequence of bondingof their atoms or the arrangement of their atoms in space, and aretermed “isomers.” Isomers that differ in the arrangement of their atomsin space are termed “stereoisomers.” Diastereomers are stereoisomerswith opposite configuration at one or more chiral centers which are notenantiomers. Stereoisomers bearing one or more asymmetric centers thatare non-superimposable mirror images of each other are termed“enantiomers.” When a compound has an asymmetric center, for example, ifa carbon atom is bonded to four different groups, a pair of enantiomersis possible. An enantiomer can be characterized by the absoluteconfiguration of its asymmetric center or centers and is described bythe R- and S-sequencing rules of Cahn, Ingold and Prelog, or by themanner in which the molecule rotates the plane of polarized light anddesignated as dextrorotatory or levorotatory (i.e., as (+) or(−)-isomers respectively). A chiral compound can exist as eitherindividual enantiomer or as a mixture thereof. A mixture containingequal proportions of the enantiomers is called a “racemic mixture”. Incertain embodiments the compound is enriched by at least about 90% byweight with a single diastereomer or enantiomer. In other embodimentsthe compound is enriched by at least about 95%, 98%, or 99% by weightwith a single diastereomer or enantiomer. The compounds of the presentdisclosure encompass racemates, diastereomers, geometric isomers,regioisomers and individual isomers thereof (e.g., separateenantiomers), and all are intended to be encompassed within the scope ofthe present disclosure.

II. Prodrug Monomers, Dimers and Conjugates

In some embodiments, the drug is a PBD monomer or a PBD dimer. In someembodiments, PDB dimers recognize and bind to specific DNA sequences.The natural product anthramycin, a PBD, was first reported in 1965(Leimgruber, et al., (1965) J. Am. Chem. Soc., 87:5793-5795; Leimgruber,et al., (1965) J. Am. Chem. Soc., 87:5791-5793). Since then, a number ofPBDs, both naturally-occurring and analogues, have been reported(Thurston, et al., (1994) Chem. Rev. 1994, 433-465 including dimers ofthe tricyclic PBD scaffold (U.S. Pat. Nos. 6,884,799; 7,049,311;7,067,511; 7,265,105; 7,511,032; 7,528,126; 7,557,099). Withoutintending to be bound by any particular theory, it is believed that thedimer structure imparts the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, In Antibiotics III. Springer-Verlag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, (1986) Acc. Chem.Res., 19:230-237). Dimeric PBD compounds bearing C2 aryl substituentshave been shown to be useful as cytotoxic agents (Hartley et al (2010)Cancer Res. 70(17):6849-6858; Antonow (2010) J. Med. Chem.53(7):2927-2941; Howard et al (2009) Bioorganic and Med. Chem. Letters19(22):6463-6466). Each reference cited in this paragraph isincorporated by reference herein in its entirety.

PBD monomers and PBD dimers within the scope of the present disclosureare known. See, for instance, US 2010/0203007, WO 2009/016516, US2009/304710, US 2010/047257, US 2009/036431, US 2011/0256157, WO2011/130598), WO 00/12507, WO 2005/085250 and WO 2005/023814, each ofwhich is incorporated by reference herein in its entirety. PBD dimerswithin the scope of the present disclosure are formed from two PBDmonomers linked at the C8 carbon atom of each PBD monomer.

A. Conjugates

In some embodiments, PBD prodrug dimer-antibody conjugates are offormula (I) comprising a first PBD prodrug monomer M1 and a secondPBD-antibody monomer M2:

M1 is a PBD monomer wherein the dashed lines represent an optionaldouble bond between one of: (i) C₁ and C₂; (ii) C₂ and C₃; and (iii) C₂and R². In some embodiments, the bond between C₁ and C₂ is a singlebond, the bond between C₂ and C₃ is a single bond, and the bond betweenC2 and R² is a double bond.

R² is selected from —H, ═CH₂, —CN, —R, ═CHR, aryl, heteroaryl, bicyclicring and heterobicyclic ring. In some embodiments, R² is ═CH₂,

R³ is hydrogen; X is selected from S, O and NH; and R¹¹ is selected from(i) H and R when X is O or NH, and (ii) H, R and O_(z)U when X is S,wherein z is 2 or 3 and U is a monovalent pharmaceutically acceptablecation.

R⁶, R and R⁹ are independently selected from H, R, OH, OR, halo, amino,nitro, SH and SR. In some embodiments, R⁶ and R⁹ are H. In someembodiments, R⁷ is OCH₃.

R¹⁰ is a prodrug moiety, described in more detail elsewhere herein,comprising (i) a glutathione-activated disulfide, (ii) aDT-diaphorase-activated quinone or (iii) a reactive oxygenspecies-activated aryl boronic acid or aryl boronic ester.

R is selected from a lower alkyl group having 1 to 10 carbon atoms andan arylalkyl group of up to 12 carbon atoms, (i) wherein the alkyl groupoptionally contains one or more carbon-carbon double or triple bonds, oran aryl group, of up to 12 carbon atoms, and (ii) wherein R isoptionally substituted by one or more halo, hydroxy, amino, or nitrogroups, and optionally contains one or more hetero atoms.

M2 is a PBD monomer wherein the dashed lines represent an optionaldouble bond between one of: (i) C_(1′) and C_(2′); (ii) C_(2′) andC_(3′); and (iii) C_(2′) and R^(2′). In some embodiments, the bondbetween C_(1′) and C_(2′) is a single bond, the bond between C_(2′) andC_(3′) is a single bond, and the bond between C_(2′) and R^(2′) is adouble bond.

R^(2′), R^(3′), R^(6′), R^(7′), R^(9′), R¹‘ and X’ correspond to, andare defined in the same way as, R², R³, R⁶, R⁷, R⁹, R¹¹ and X,respectively.

L is a self-immolative linker comprising at least one of a disulfidemoiety, a peptide moiety and a peptidomimetic moiety. In someembodiments, the linker comprises a disulfide moiety or a peptidemoiety.

Each asterisk independently represents a chiral center of racemic orundefined stereochemistry.

M1 and M2 are bound at the C8 position by a moiety -Q-T-Q′-, wherein Qand Q′ are independently selected from O, NH and S, and wherein T is anoptionally substituted C₁₋₁₂ alkylene group that is further optionallyinterrupted by one or more heteroatoms and/or aromatic rings. In someembodiments, Q and Q′ are O, and T is C₃ alkylene or C₅ alkylene.

Ab is an antibody as defined elsewhere herein. In some embodiments, theantibody comprises at least one cysteine sulfhydryl moiety, wherein theantibody binds to one or more tumor-associated antigens or cell-surfacereceptors selected from: (1) BMPR1B (bone morphogenetic proteinreceptor-type IB); (2) E16 (LAT1, SLC7A5); (3) STEAP1 (six transmembraneepithelial antigen of prostate); (4) MUC16 (0772P, CA125); (5) MPF (MPF,MSLN, SMR, megakaryocyte potentiating factor, mesothelin); (6) Napi2b(NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate),member 2, type II sodium-dependent phosphate transporter 3b); (7) Sema5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5b Hlog,sema domain, seven thrombospondin repeats (type 1 and type 1-like),transmembrane domain (TM) and short cytoplasmic domain, (semaphorin)5B); (8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12,RIKEN cDNA 2700050C12 gene); (9) ETBR (Endothelin type B receptor); (10)MSG783 (RNF124, hypothetical protein FLJ20315); (11) STEAP2 (HGNC_8639,IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostate cancer associated gene1, prostate cancer associated protein 1, six transmembrane epithelialantigen of prostate 2, six transmembrane prostate protein); (12) TrpM4(BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cationchannel, subfamily M, member 4); (13) CRIPTO (CR, CR1, CRGF, CRIPTO,TDGF1, teratocarcinoma-derived growth factor); (14) CD21 (CR2(Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) or Hs73792); (15) CD79b (CD79β, CD790, IGb (immunoglobulin-associated beta),B29); (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containingphosphatase anchor protein 1a), SPAP1B, SPAP1C); (17) HER2; (18) NCA;(19) MDP; (20) IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24)PSCA; (25) GEDA; (26) BAFF-R (B cell—activating factor receptor, BLySreceptor 3, BR3); (27) CD22 (B-cell receptor CD22-B isoform); (28) CD79a(CD79A, CD79α, immunoglobulin-associated alpha); (29) CXCR5 (Burkitt'slymphoma receptor 1); (30) HLA-DOB (Beta subunit of MHC class IImolecule (Ia antigen)); (31) P2X5 (Purinergic receptor P2X ligand-gatedion channel 5); (32) CD72 (B-cell differentiation antigen CD72, Lyb-2);(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family); (34) FcRH1 (Fc receptor-like protein1); (35) FcRH5 (IRTA2, Immunoglobulin superfamily receptor translocationassociated 2); (36) TENB2 (putative transmembrane proteoglycan); (37)PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); (38) TMEFF1(transmembrane protein with EGF-like and two follistatin-like domains 1;Tomoregulin-1); (39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1;GDNFR; GDNFRA; RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); (40)Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E, SCA-2, TSA-1);(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); (42) Ly6G6D(lymphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1); (43) LGR5(leucine-rich repeat-containing G protein-coupled receptor 5; GPR49,GPR67); (44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC;CDHF12; Hs.168114; RET51; RET-ELE1); (45) LY6K (lymphocyte antigen 6complex, locus K; LY6K; HSJ001348; FLJ35226); (46) GPR19 (Gprotein-coupled receptor 19; Mm.4787); (47) GPR54 (KISS1 receptor;KISS1R; GPR54; HOT7T175; AXOR12); (48) ASPHD1 (aspartatebeta-hydroxylase domain containing 1; LOC253982); (49) Tyrosinase (TYR;OCAIA; OCA1A; tyrosinase; SHEP3); (50) TMEM118 (ring finger protein,transmembrane 2; RNFT2; FLJ14627); (51) GPR172A (G protein-coupledreceptor 172A; GPCR41; FLJ11856; D15Ertd747e); (52) CD33; and (53)CLL-1. In some embodiments, the antibody is engineered for conjugation.In some embodiments, the antibody is a cysteine-engineered antibodycomprising LC K149C, HC A118C, HC A140C or LC V205C as the site oflinker conjugation. In some embodiments, the antibody orcysteine-engineered antibody is selected from anti-HER2, anti-CD22,anti-CD33, anti-Napi2b, anti-Ly6E, and anti-CLL-1.

The integer p is 1, 2, 3, 4, 5, 6, 7 or 8. In some embodiments, p is 1,2, 3 or 4. In some embodiments, a composition comprising a mixture ofPBD prodrug dimer-antibody conjugates is provided wherein the averagedrug loading per antibody in the mixture of conjugate compounds is about2 to about 5.

In some embodiments, R⁷ and R^(7′) are —OCH₃; R³, R^(3′), R⁶, R^(6′), R⁹and R^(9′) are H; and R² and R^(2′) are ═CH₂,

In some embodiments, the bond between C₁ and C₂ of M1 is a single bond;the bond between C₂ and C₃ of M1 is a single bond; the bond betweenC_(1′) and C_(2′) of M2 is a single bond; the bond between C_(2′) andC_(3′) of M2 is a single bond; C₃ of M1 is substituted with two R³groups, each of which is H; C_(3′) of M2 is substituted with two R^(3′)groups, each of which is H; the bond between C₂ and R² of M1 is a doublebond; and the bond between C_(2′) and R^(2′) of M2 is a double bond.

In some embodiments, the PBD prodrug dimer-antibody conjugate compoundis of formula (Ia):

wherein R¹⁰, L, p and Ab are as defined elsewhere herein.

B. Monomers

In some embodiments, PBD monomer compounds are of formula (II):

wherein R², R³, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, X, *, and the bonding schemein pyrrolidine ring A are as described elsewhere herein in connectionwith the PBD dimers.

In some embodiments, the PBD monomer compound is of formula (IIa):

wherein R¹⁰ is as defined elsewhere herein.

C. Dimers

In some embodiments, PBD prodrug dimer compounds are of formula (VIII)comprising a first PBD prodrug monomer M1 and a second PBD monomer M2:

wherein R², R^(2′), R³, R^(3′), R⁶, R^(6′), R⁷, R^(7′), R⁹, R^(9′), R¹⁰,R¹¹, R^(11′), X, X′, Q, Q′, T, *, and the bonding schemes in pyrrolidinerings A and B are as described elsewhere herein in connection with thePBD dimers.

In some embodiments, R¹² is absent and the bond between N10′ and C_(11′)is a double bond. In some embodiments, R¹² is selected from —C(O)O-L and—C(O)O—R¹⁰ where R¹⁰ is a prodrug moiety as described elsewhere herein.L is as defined elsewhere herein.

In some embodiments, the PBD prodrug dimer compounds are of formula(VIIIa):

wherein R¹⁰ is as defined elsewhere herein.

In some embodiments, the PBD prodrug dimer compounds are of formula(VIIb):

wherein R¹⁰ and L are as defined elsewhere herein.

III. Prodrug Protecting Group-Trigger

Prodrug protecting groups comprising a trigger within the scope of thepresent disclosure include disulfides, cyclic diones, aryl boronic acidsand aryl boronic esters. Prodrug protecting groups comprising a triggerare conjugated to PBDs at the N10 position by a carbamate moiety. Theprotecting group is enzymatically or chemically cleaved to generate theactive drug by the application of stimulus, such as an enzyme (e.g.,DTD), ROS or GSH.

A. Disulfide Protecting Group-Triggers

Disulfide protection group-trigger R¹⁰ moieties of the presentdisclosure are of the general formula (V):

wherein the wavy line indicates the point of attachment to the PBD N10position

R⁵⁰ is selected from optionally substituted C₁₋₈ alkyl or C₂₋₆ alkyl,optionally substituted C₁₋₈ or C₂₋₆ heteroalkyl, optionally substitutedcycloalkyl comprising from 2 to 6 carbon atoms, and optionallysubstituted heterocycloalkyl comprising from 2 to 6 carbon atoms. Insome particular embodiments, R⁵⁰ is selected from —CH₂—CH₃, —CH(CH₃)₂,—C(CH₃)₃, —CH₂—CH₂OH, —CH₂—CH₂—C(O)OH, —CH₂—CH₂—O—CH₃, and a 3- to6-membered cycloalkyl or a heterocycloalkyl. In some embodiments,cycloalkyl and heterocycloalkyl R⁵⁰ moieties are selected from:

In some embodiments, R⁵⁰ is selected from CH₃CH₂—, (CH₃)₂CH— and(CH₃)₃C—.

R⁵¹ is optionally substituted C₂ alkylene or optionally substitutedbenzylene. In some such embodiments, R⁵¹ is of the formula:

In some embodiments, R⁶¹ and R⁶² are independently selected from H andoptionally substituted C₁₋₆ alkyl, and optionally substituted C₁₋₆heteroalkyl. In some particular embodiments, R⁶¹ and R⁶² areindependently selected from H and optionally substituted C₁₋₄ alkyl, andoptionally substituted C₁₋₄ ether or tertiary amine. In some otherparticular embodiments, one of R⁶¹ and R⁶² is H. In yet other particularembodiments, one of R⁶¹ and R⁶² is H and the other of R⁶¹ and R⁶² is—CH₃, C₁₋₄ ether or C tertiary amine. In some other particularembodiments, R⁶¹ and R⁶² are each H or R⁶¹ and R⁶² are each CH₃.

In some embodiments, R⁶¹ and R⁶² together with the carbon atom to whichthey are bound form an optionally substituted cycloalkyl, optionallysubstituted heterocycloalkyl, optionally substituted aryl or optionallysubstituted heteroaryl moiety, each ring substitution comprising from 2to 6 carbon atoms.

In some embodiments, R⁶³ and R⁶⁴ are independently selected from H andCH₃. In some other particular embodiments, R⁶³ and R⁶⁴ are H.

A non-limiting listing of exemplary R⁵¹ moieties is as follows:

In some alternative embodiments, R⁵¹ is an arylalkyl. In one suchembodiment, R⁵¹ is:

A non-limiting listing of exemplary disulfide protection group-triggermoieties is as follows:

In accordance with the present disclosure and based on in vitroexperimental evidence to-date, it has been discovered that disulfideprotection group-triggers of the present disclosure are cleavedintracellularly in proliferating cells, such as cancer cells, expressingelevated GSH, and are generally stable in non-proliferating cellsexpressing normal GSH levels as well as in whole blood/plasma. Moreparticularly, blood concentration of GSH is known to be very low, suchas in the micromolar range, whereas intracellular GSH concentration istypically up to three orders of magnitude greater, such as in themillimolar range. It is further believed that GSH concentration incancer cells is even greater, due to increased activity of reductiveenzymes.

It has been further discovered that differences between intracellularreduction potential (expressed in mV) between proliferating andnon-proliferating cells may be exploited to effect disulfide triggeractivation and drug release in proliferating cells, while providing forprodrug stability in non-proliferating cells, whole blood and plasma.More particularly, it is believed that the ratio of reduced GSH tooxidized GSH (also termed GSH disulfide or “GSSG”) in the GSH/GSSG redoxcouple is correlated with reduction potential, typically expressed inmV. Certain GSH/GSSG ratios are further believed to be characteristic ofproliferating cells. Reduction potential negativity increases as theratio of GSH/GSSG increases (i.e., as the relative concentration of GSHincreases). Typical GSH/GSSG reduction potentials are presented in thetable below:

Cytoplasm Reduction Potential (mV) Blood/plasma −140 Proliferating cell−260 to −230 Growth arrest cell −220 to −190 Apoptotic cell −170 to −150

The reduction potential of the cysteine (Cys) and cysteine disulfide(CySS) redox couple may also effect intracellular drug release fromdisulfide prodrugs where the blood/plasma Cys/CySS reduction potentialis typically −80 mV to 0 mV and the Cys/CySS reduction potential in thecytoplasm is typically about −160 mV.

B. Cyclic Dione Protecting Group-Triggers

In some embodiments, R¹⁰ cyclic dione protecting group-triggers are 1,4-or 1,2-quinones of the general formulae:

A, D, E, G and J are independently selected from C and N wherein N is asecondary amine, a tertiary amine or an imine (═N—). Each m isindependently selected from 0 and 1. The dashed lines represent anoptional double bond between either E-D or D-A. R^(A), R^(D), R^(E),R^(G) and R^(J), when present, are independently selected from H, OH,optionally substituted C₁₋₄ alkyl or heteroalkyl, C₁₋₄ alkoxy orheteroalkoxy, and halogen, with the proviso that at least one of R^(A),R^(D), R^(E), R^(G) and R^(J) is a C₁₋₄ optionally substituted alkyl orheteroalkyl linker that is covalently bound to the oxygen atom of acarbamate moiety at the PBD N10 position. In some embodiments, one ofR^(A), R^(D) and R^(E) is the C₁₋₄ optionally substituted alkyl orheteroalkyl linker. In some other embodiments, one of A-(R^(A))_(m),D-(R^(D))_(m) and E-(R^(E))_(m) is

In some other embodiments, A-(R^(A))_(m) is

D is C and R^(D) is a C₁ linker, E-(R^(E))_(m) is

where the bond between D and E is a double bond. In other embodiments, Gand J are C (carbon). In other embodiments, G and J are C, and one ofR^(G) and R^(J) is —O—CH₃. In some embodiments, the cyclic dione is a1,4-cyclic dione.

In some embodiments, the cyclic dione prodrug moiety is a quinoneselected from the following:

wherein the hydroxyl moiety provides the point of attachment to the PBD.

In some particular embodiments, the cyclic dione is an indole dione ofone of the following formulae:

wherein the wavy line indicates the point of attachment to the oxygenatom at the PBD N10 position

In some embodiments, R¹⁰ cyclic dione protecting group-triggers are 1,4-or 1,2-cyclic diones of the following formulae:

A, D, E, F, G and J are independently selected from C and N, wherein atleast one of A, D, E and F is C and at least one of G and J is C, andwherein N is a secondary anime, a tertiary amine or an imine (═N—). Eachn is independently selected from 0 and 1. The dashed lines representoptional double bonds. R^(A), R^(D), R^(E), R^(F), R^(G) and R^(J), whenpresent, are independently selected from H, OH and optionallysubstituted C₁₋₄ alkyl or heteroalkyl, C₁₋₄ alkoxy or heteroalkoxy, andhalogen, with the proviso that at least one of R^(A), R^(D), R^(E),R^(F), R^(G) and R^(J) is present and is a C₁₋₄ optionally substitutedalkyl or heteroalkyl linker that is covalently bound to the oxygen atomof a carbamate moiety at the PBD N10 position. In some embodiments, oneof R^(A), R^(D), R^(E) and R^(F) is the C₁₋₄ optionally substitutedalkyl or heteroalkyl linker. In some embodiments, D-R^(D) or E-R^(E) isa moiety having a carbon atom bound to the C₁₋₄ optionally substitutedalkyl or heteroalkyl linker, and at least one of A, F and the other of Dand E is N. In some embodiments, one of R^(A), R^(E) and R^(F) areindependently selected from H, optionally substituted C₁₋₄ alkyl orheteroalkyl and optionally substituted C₁₋₄ alkoxy or heteroalkoxy, andwherein D is C and R^(D) is a C₁ linker. In some other embodiments, atleast one of G-(R^(G))_(n) and J-(R^(J))_(n) is

In some other embodiments, one of G-R^(G) and J-R^(J) is C—O—CH₃, theother of G-R^(G) and J-R^(J) is CH, where the bond between G and J is adouble bond. In some other embodiments, the ring formed from A, D, E andF is unsaturated or partially saturated. In other embodiments, the ringformed from A, B, D and E is unsaturated or partially saturated.

Prodrugs having a cyclic dione protecting group-trigger may be activatedwith a DTD two-electron reducing enzyme that is believed to beover-expressed in many human tumors and in the endothelial cells ofblood vessels. DTD is a NAD(P)H:quinone oxidoreductase type I (NQO1)enzyme (EC 1.6.99.2) that catalyzes the direct two-electron transfer ofquinones using NADH or NADPH as a cofactor (see, e.g., Mendoza, et al.,“Human NAD(P)H:quinone oxidoreductase type I (HNQO1) activation ofquinone propionic acid trigger groups”, Biochemistry, 2012 Oct. 9;51(40): 8014-8026, incorporated by reference herein). It is believedthat DTD is a prodrug activator under both aerobic and hypoxicconditions.

Human breast and lung cancers are known to express high DTD levels. Forinstance, NQO1 expression in nRPKM (where nRPKM refers to normalizedreads per Kb of transcript length per million mapped read) is on theorder of 30 to 2000 as compared to blood and lymph cancers thattypically have nRPKM values in the range of from about 0.5 to about 20.As disclosed in the table below, DTD is over-expressed in many cancersrelative to normal tissue (see S. Danson, et al., “DT-diaphorase: atarget for new anticancer drugs”, Cancer Treatment Reviews (2004) 30,437-449) where “NS” refers to not significant:

Cell DTD ratio to normal tissue Human colon carcinoma primary 2.5 to 3.9Human colon carcinoma metastasis 47 Human breast carcinoma NS to 9.5Human NSCLC  8.2 to 19.2 Human liver carcinoma 3.8 to 50 

Without being bound to any particular theory, the intracellular prodrugrelease mechanism is believed to generally proceed according to thefollowing mechanism, as represented by one quinone species, wherein theprodrug quinone trigger activation and drug release is mediated by DTDtwo-electron reduction:

C. Aryl Boronic Acid and Aryl Boronic Ester Protecting Group-Triggers

Aryl boronic acid and aryl boronic ester protection group-trigger R¹⁰moieties of the present disclosure are of the general formula (IVa):

wherein the wavy line indicates the point of attachment to the oxygenatom at the PBD N10 position (i.e., —O—C(O)—N₁₀<PBD). R²⁰ and R²¹ areindependently selected from H, optionally substituted alkyl orheteroalkyl, optionally substituted cycloalkyl or heterocycloalkyl, andoptionally substituted aryl or heteroaryl. Alternatively, R²⁰ and R²¹together are an optionally substituted moiety —(CH₂)_(n)—, wherein n is2 or 3, said moiety together with the O atoms to which they are attachedand the B atom form a heterocycloalkyl ring. The heterocycloalkyl ringmay optionally comprise a fused heteroalkyl ring, a fused aryl ring or afused heteroaryl ring. The wavy line indicates the point of attachmentto the PBD N10 position.

In some embodiments, aryl boronic acid and aryl boronic ester triggersare of the formulae:

R³⁰, R³¹, R³², R³³, R⁴⁰, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ are independentlyselected from H, halogen, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,—SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, optionallysubstituted C₁₋₈ alkyl or heteroalkyl, optionally substituted cycloalkylor heterocycloalkyl comprising from 2 to 7 carbon atoms, optionallysubstituted aryl or heteroaryl. In some embodiments, (i) one of R³⁰ orR³¹ and one of R³² or R³³, (ii) one of R⁴⁰ or R⁴¹ and one of R⁴² or R⁴³,and/or (iii) one of R⁴² or R⁴³ and one of R⁴⁴ or R⁴⁵ form an optionallysubstituted fused cycloalkyl ring, fused heterocycloalkyl ring, fusedaryl ring or fused heteroaryl ring having from 2 to 7 carbon atoms. Thewavy line indicates the point of attachment to the PBD N10 position.

A non-limiting listing of exemplary aryl boronic acid and aryl boronicester protecting group-triggers is as follows:

In one embodiment, the protecting group-trigger is aryl boronic acid,i.e., where R²⁰ and R²¹ are H.

Prodrugs having an aryl boronic acid or an aryl boronic ester protectinggroup-trigger may be activated with a ROS, such as H₂O₂. (See, forinstance, Kuang, Y., et al., “Hydrogen Peroxide Inducible DNACross-Linking Agents: Targeted Anticancer Prodrugs”, J. Am. Chem. Soc.(2011), 133(48), 19278-19281; Peng, X., et al., “ROS-activatedanticancer prodrugs: a new strategy for tumor-specific damage”, TherDeliv. (2012), 3(7), 823-833; and Chen, W., et al., “Reactive OxygenSpecies (ROS) Inducible DNA Cross-Linking Agents and Their Effect onCancer Cells and Normal Lymphocytes”, J. Med. Chem. (2014), 57,4498-4510, each of which is incorporated by reference herein in itsentirety.)

Cancer cells are believed to exhibit increased oxidative stress ascompared to normal, non-cancerous, cells and are believed to haveincreased cellular concentrations of ROS, such as H₂O₂, wherein H₂O₂concentration may be elevated in cancer cells by ten-fold, such as up to0.5 nmol/10⁴ cells/h. (See, e.g., Peng; Chen; Zieba, M., et al.,“Comparison of hydrogen peroxide generation and the content of lipidperoxidation products in lung cancer tissue and pulmonary parenchyma”,Respiratory Medicine (2000), 94, 800-805; Szatrowski, T. et al.,“Production of Large Amounts of Hydrogen Peroxide in Human Tumor Cells”,Cancer Research (1991), 51, 794-798, each of which is incorporated byreference herein.) It is believed that the high ROS concentration incancer cells, and concomitant ROS signaling, is a major factor in tumorformation, development, proliferation and survival through DNA mutation,metastasis, angiogenesis and reduced sensitivity to therapeutic agents(see, e.g., Peng).

Without being bound to any particular theory, the ROS-activatedintracellular prodrug release mechanism is believed to proceed accordingto the following mechanism:

IV. Linkers

The linkers of the present disclosure are bifunctional chemical moietiesthat are capable of covalently linking together an antibody and a drug(“D”) into a tripartite molecule. Linkers within the scope of thepresent disclosure are not narrowly limited and are of the generalstructure:Ab-[Ab connection]-[Ab spacer]_(opt)-[Trigger]_(opt)-[D spacer]_(opt)-[Dconnection]-Dand comprise an Ab connection, an optional Ab spacer unit, an optionalimmolative (trigger) unit, and optional drug spacer, and a drugconnection.

In some embodiments, the linker comprises a self-immolative moiety(trigger). Non-limiting examples of self-immolative moieties within thescope of the present disclosure include peptides, peptidomimetics anddisulfides.

In some embodiments, the linker comprises an immolative peptide unitallowing for enzymatic cleavage of the linker, such as by a protease,thereby facilitating release of the drug from the immunoconjugate uponexposure to intracellular proteases, such as lysosomal enzymes (Doroninaet al. (2003) Nat. Biotechnol. 21:778-784). Exemplary peptide unitsinclude, but are not limited to, dipeptides, tripeptides, tetrapeptides,and pentapeptides. Exemplary dipeptides include, but are not limited to,valine-citrulline (vc or val-cit), valine-alanine (va or val-ala),alanine-phenylalanine (af or ala-phe); phenylalanine-lysine (fk orphe-lys); phenylalanine-homolysine (phe-homolys); andN-methyl-valine-citrulline (Me-val-cit). Exemplary tripeptides include,but are not limited to, glycine-valine-citrulline (gly-val-cit) andglycine-glycine-glycine (gly-gly-gly). A peptide unit may comprise aminoacid residues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Peptideunits can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, cathepsinB, C and D, or a plasmin protease.

In some embodiments, the linker comprises an immolative peptidomimeticunit allowing for cleaving of the linker. Exemplary peptidomimetic unitsinclude, but are not limited to, triazoles,cyclobutane-1-1-dicarbaldehyde,cyclobutane-1-1-dicarbaldehyde-citrulline, alkenes, haloalkenes, andisoxazoles. Some peptidomimentic unit examples include the followingwhere the wavy line at the left side of the peptidomimetic unit is thepoint of connection to a spacer or an antibody connection moiety and thewavy line at the right side of the peptidomimetic unit is the point ofconnection to a spacer or a drug connection moiety:

Examples of some Ab-[peptidomimetic linker unit]-Drug groups within thescope of the present disclosure are as follows, where “AA” refers to anamino acid, where AA1 and AA2 may be the same, or different, naturallyoccurring or non-naturally occurring amino acid:

In some embodiments, the linker comprises an immolative disulfide unitallowing for cleavage of the linker. Disulfide linkers generally are ofthe formula:

wherein: S_(C) is an antibody cysteine sulfur atom; R⁷⁰ and R⁷¹ areindependently selected from H and C₁₋₃ alkyl, wherein only one of R⁷⁰and R⁷¹ can be H, or R⁷⁰ and R⁷¹ together with the carbon atom to whichthey are bound form a four- to six-membered ring optionally comprisingan oxygen heteroatom; and, the wavy line indicates the point ofattachment to the oxygen atom of the carbamate moiety at a PBD N10position. In some other embodiments, R⁷⁰ and R⁷¹ are independentlyselected from H, —CH₃ and —CH₂CH₃, wherein only one of R⁷⁰ and R⁷¹ canbe H, or R⁷⁰ and R⁷¹ together with the carbon atom to which they arebound form a ring selected from cyclobutyl, cyclopentyl, cyclohexyl,tetrahydrofuran and tetrahydropyran.

In some embodiments, the linker may comprise a spacer unit. In someembodiments, the spacer unit is p-aminobenzyloxycarbonyl (PAB). In somesuch embodiments, a p-aminobenzyl alcohol spacer unit is attached to anamino acid unit via an amide bond, and a carbamate, methylcarbamate, orcarbonate connection is made between the benzyl alcohol and the drug(Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103).In other embodiments, the linker-antibody moiety is attached to theoxygen atom of the carbamate moiety at the PBD N10 position as follows:

In some other embodiments, spacers include, but are not limited to,aromatic compounds that are electronically similar to the PAB group,such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078;Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- orpara-aminobenzylacetals.

In some embodiments, spacers can be used that undergo cyclization uponamide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ringsystems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and2-aminophenylpropionic acid amides (Amsberry, et al (1990) J. Org. Chem.55:5867). Linkage of a drug to the alpha-carbon of a glycine residue isanother example of a spacer that may be useful (Kingsbury et al (1984)J. Med. Chem. 27:1447). In some aspects, the spacer unit isself-immolative.

The linker comprises a reactive group suitable for covalent conjugationto an antibody. In some embodiments, the antibody comprises at least onereactive sulfhydryl moiety and the linker comprises a reactive sulfuratom, a maleimide, a bromacetamide, and iodoacetamide, or an alkene,wherein the antibody is conjugated to the linker by a covalent bondformed by the reaction of the antibody reactive sulfhydryl with thelinker reactive sulfur atom, maleimide, bromacetamide, iodoacetamide, oralkene according to methods known to those skilled in the art.

Non-limiting examples of schemes for conjugating an antibody having areactive sulfhydryl moiety to a Drug-Linker moiety are indicated asfollows:

Examples of some particular Drug-[L] conjugates are as follows, where xis from 1 to 8 and where [Conj] refers to a reactive group as describedelsewhere herein:

In embodiments comprising an immolative disulfide, conjugation to the Abmay be done according to the methods described in Application No.PCT/CN2015/092084, incorporated herein by reference in its entirety. Ingeneral, an activated leaving group-disulfide-drug compound of thefollowing formula:

is contacted with an antibody having at least one sulfhydryl moiety, theleaving group (X_(L)) is displaced, and the sulfur atom is covalentlybound to the sulfhydryl sulfur atom to form a disulfide. In the aboveformula: X_(L) is a thiol leaving group; the leaving group and thelinker are bound via a disulfide bond; R⁷⁰ and R⁷¹ are as definedelsewhere herein; and, Sp is an optional spacer as described elsewhereherein, wherein n is 0 or 1. The linker may be considered to be ahindered linker because only one of R⁷⁰ and R⁷¹ may be H.

In some embodiments, the leaving group may suitably be selected from thefollowing:

In such embodiments, the wavy lines indicate the point of attachment ofthe leaving group to the hindered linker S atom thereby forming adisulfide bond. X¹, X², X³, X⁴ and X⁵ are independently C, N, S or O,provided at least one of X¹ to X⁵ is N, the dashed lines representoptional double bonds, and A denotes a six-membered ring. Y¹, Y² Y³ andY⁴ are independently C, N, S or O, provided at least one of Y¹ to Y⁴ isN, the dashed lines represent optional double bonds, and B denotes afive-membered ring. Z¹, Z², Z³, Z⁴, Z⁵ and Z⁶ are independently C, N, Sor O, provided at least one of Z¹ and Z² is N, the dashed linesrepresent optional double bonds, C denotes a six-membered ring, and Ddenotes a fused five-membered ring. Each R³ is independently selectedfrom —NO₂, —NH₂, —C(O)OH, R⁵S(O)(O)—, —C(O)N(R⁵)(R⁵), —Cl, —F, —CN and—Br. Each R⁵ is independently selected from H, optionally substitutedC₁₋₆ hydrocarbyl, optionally substituted C₅₋₆ carbocycle, and optionallysubstituted C₅₋₆ heterocycle, and q is 1, 2 or 3. Each carbon atom inthe ring structure of leaving group 1, leaving group 2, leaving group 3and/or leaving group 4 is optionally substituted with R⁵. Each nitrogenatom in the ring structure of leaving group 1, leaving group 2, leavinggroup 3 and/or leaving group 4 is optionally substituted with R⁵ to forma tertiary amine or a quaternary amine.

In some particular such embodiments, X¹, X², X³, X⁴ and X⁵ areindependently C or N, no more than two of X¹ to X⁵ are N, and ring A isunsaturated. In other particular embodiments, Y¹, Y² Y³ and Y⁴ areindependently C or N, and B ring is unsaturated. In yet other particularembodiments, Z¹ is N, Z² is selected from N, S and O, Z³ to Z⁶ areselected from C and N, no more than two of Z³ to Z⁶ are N, and ring C isunsaturated. In still other particular embodiments, Each R³ isindependently selected from —NO₂, —NH₂, —C(O)OH, H₃CS(O)(O)— and—C(O)N(CH₃)₂.

In some embodiments, the leaving group is

wherein the wavy line indicates the point of attachment of the leavinggroup to the hindered linker S atom thereby forming a disulfide bond. Insome particular embodiments C₁₋₄ alkyl is methyl.

Some examples of leaving groups of the present disclosure areillustrated below:

Examples of hindered disulfide linkers are as follows where the wavyline at the sulfur atom refers to the point of attachment to a leavinggroup as defined elsewhere herein and wherein the wavy line at thecarbonyl moiety refers to the point of attachment to a PBD N10 atom:

V. Antibodies

The antibodies of the present disclosure are any cell-targeting biologiccompound that binds to one or more tumor-associated antigens or tocell-surface receptors, the antibodies comprising at least one reactivecysteine sulfhydryl moiety suitable for conjugation to a linker.

Certain types of cells, such as cancer cells, express surface molecules(antigens) that are unique as compared to surrounding tissue. Celltargeting moieties that bind to these surface molecules enable thetargeted delivery of a drug described elsewhere herein specifically tothe target cells. For instance and without limitation, a cell targetingmoiety may bind to and be internalized by a lung, breast, brain,prostate, spleen, pancreatic, cervical, ovarian, head and neck,esophageal, liver, skin, kidney, leukemia, bone, testicular, colon orbladder cell.

A. Tumor Associated Antigens

In some particular embodiments of the disclosure, the target cells arecancer cells that express tumor-associated antigens (TAA) or thatcomprise cell-surface receptors. Tumor-associated antigens are known inthe art, and can be prepared for use in generating antibodies usingmethods and information which are well known in the art. In attempts todiscover effective cellular targets for cancer diagnosis and therapy,researchers have sought to identify transmembrane or otherwisetumor-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of tumor-associated antigens TAA include, but are not limitedto, TAA (1)-(53) listed herein. For convenience, information relating tothese antigens, all of which are known in the art, is listed herein andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(53) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

(1) BMPR1B (bone morphogenetic protein receptor-type IB, Genbankaccession no. NM_001203) ten Dijke, P., et al. Science 264(5155):101-104 (1994), Oncogene 14 (11):1377-1382 (1997)); WO2004063362(Claim 2); WO2003042661 (Claim 12); US2003134790-A1 (Page 38-39);WO2002102235 (Claim 13; Page 296); WO2003055443 (Page 91-92);WO200299122 (Example 2; Page 528-530); WO2003029421 (Claim 6);WO2003024392 (Claim 2; FIG. 112); WO200298358 (Claim 1; Page 183);WO200254940 (Page 100-101); WO200259377(Page 349-350); WO200230268(Claim 27; Page 376); WO200148204 (Example; FIG. 4) NP_001194 bonemorphogenetic protein receptor, typeIB/pid=NP_001194.1—Cross-references: MIM:603248; NP_001194.1; AY065994.

(2) E16 (LAT1, SLC7A5, Genbank accession no. NM_003486) Biochem.Biophys. Res. Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291(1998), Gaugitsch, H. W., et al. (1992) J. Biol. Chem. 267 (16):11267-11273); WO2004048938 (Example 2); WO2004032842 (Example IV);WO2003042661 (Claim 12); WO2003016475 (Claim 1); WO200278524 (Example2); WO200299074 (Claim 19; Page 127-129); WO200286443 (Claim 27; Pages222, 393); WO2003003906 (Claim 10; Page 293); WO200264798 (Claim 33;Page 93-95); WO200014228 (Claim 5; Page 133-136); US2003224454 (FIG. 3);WO2003025138 (Claim 12; Page 150); NP_003477 solute carrier family 7(cationic amino acid transporter, y+ system), member5/pid=NP_003477.3—Homo sapiens Cross-references: MIM:600182;NP_003477.3; NM_015923; NM_003486_1.

(3) STEAP1 (six transmembrane epithelial antigen of prostate, Genbankaccession no. NM_012449) Cancer Res. 61 (15), 5857-5860 (2001), Hubert,R. S., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528);WO2004065577 (Claim 6); WO2004027049 (FIG. 1L); EP1394274 (Example 11);WO2004016225 (Claim 2); WO2003042661 (Claim 12); US2003157089 (Example5); US2003185830 (Example 5); US2003064397 (FIG. 2); WO200289747(Example 5; Page 618-619); WO2003022995 (Example 9; FIG. 13A, Example53; Page 173, Example 2; FIG. 2A); NP_036581 six transmembraneepithelial antigen of the prostate Cross-references: MIM:604415;NP_036581.1; NM_012449_1.

(4) 0772P (CA125, MUC16, Genbank accession no. AF361486) J. Biol. Chem.276 (29):27371-27375 (2001)); WO2004045553 (Claim 14); WO200292836(Claim 6; FIG. 12); WO200283866 (Claim 15; Page 116-121); US2003124140(Example 16); U.S. Pat. No. 798,959. Cross-references: GI:34501467;AAK74120.3; AF361486_1.

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin,Genbank accession no. NM_005823) Yamaguchi, N., et al. Biol. Chem. 269(2), 805-808 (1994), Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536(1999), Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996), J. Biol.Chem. 270 (37):21984-21990 (1995)); WO2003101283 (Claim 14);(WO2002102235 (Claim 13; Page 287-288); WO2002101075 (Claim 4; Page308-309); WO200271928 (Page 320-321); WO9410312 (Page 52-57);Cross-references: MIM:601051; NP_005814.2; NM_005823_1.

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b,Genbank accession no. NM_006424) J. Biol. Chem. 277 (22):19665-19672(2002), Genomics 62 (2):281-284 (1999), Feild, J. A., et al. (1999)Biochem. Biophys. Res. Commun. 258 (3):578-582); WO2004022778 (Claim 2);EP1394274 (Example 11); WO2002102235 (Claim 13; Page 326); EP875569(Claim 1; Page 17-19); WO200157188 (Claim 20; Page 329); WO2004032842(Example IV); WO200175177 (Claim 24; Page 139-140); Cross-references:MIM:604217; NP_006415.1; NM_006424_1.

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B, Genbank accession no. AB040878) Nagase T., et al.(2000) DNA Res. 7 (2):143-150); WO2004000997 (Claim 1); WO2003003984(Claim 1); WO200206339 (Claim 1; Page 50); WO200188133 (Claim 1; Page41-43, 48-58); WO2003054152 (Claim 20); WO2003101400 (Claim 11);Accession: Q9P283; EMBL; AB040878; BAA95969.1. Genew; HGNC:10737.

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene, Genbank accession no. AY358628); Ross et al.(2002) Cancer Res. 62:2546-2553; US2003129192 (Claim 2); US2004044180(Claim 12); US2004044179 (Claim 11); US2003096961 (Claim 11);US2003232056 (Example 5); WO2003105758 (Claim 12); US2003206918 (Example5); EP1347046 (Claim 1); WO2003025148 (Claim 20); Cross-references:GI:37182378; AAQ88991.1; AY358628_1.

(9) ETBR (Endothelin type B receptor, Genbank accession no. AY275463);Nakamuta M., et al. Biochem. Biophys. Res. Commun. 177, 34-39, 1991;Ogawa Y., et al. Biochem. Biophys. Res. Commun. 178, 248-255, 1991; AraiH., et al. Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al. J. Biol.Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al. Biochem.Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N. A., et al. J.Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al. J. Cardiovasc.Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al. Gene 228, 43-49, 1999;Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A. 99, 16899-16903,2002; Bourgeois C., et al. J. Clin. Endocrinol. Metab. 82, 3116-3123,1997; Okamoto Y., et al. Biol. Chem. 272, 21589-21596, 1997; Verheij J.B., et al. Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., etal. Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al.Cell 79, 1257-1266, 1994; Attie T., et al., Hum. Mol. Genet. 4,2407-2409, 1995; Auricchio A., et al. Hum. Mol. Genet. 5:351-354, 1996;Amiel J., et al. Hum. Mol. Genet. 5, 355-357, 1996; Hofstra R. M. W., etal. Nat. Genet. 12, 445-447, 1996; Svensson P. J., et al. Hum. Genet.103, 145-148, 1998; Fuchs S., et al. Mol. Med. 7, 115-124, 2001;Pingault V., et al. (2002) Hum. Genet. 111, 198-206; WO2004045516 (Claim1); WO2004048938 (Example 2); WO2004040000 (Claim 151); WO2003087768(Claim 1); WO2003016475 (Claim 1); WO2003016475 (Claim 1); WO200261087(FIG. 1); WO2003016494 (FIG. 6); WO2003025138 (Claim 12; Page 144);WO200198351 (Claim 1; Page 124-125); EP522868 (Claim 8; FIG. 2);WO200177172 (Claim 1; Page 297-299); US2003109676; U.S. Pat. No.6,518,404 (FIG. 3); U.S. Pat. No. 5,773,223 (Claim 1a; Col 31-34);WO2004001004.

(10) MSG783 (RNF124, hypothetical protein FLJ20315, Genbank accessionno. NM_017763); WO2003104275 (Claim 1); WO2004046342 (Example 2);WO2003042661 (Claim 12); WO2003083074 (Claim 14; Page 61); WO2003018621(Claim 1); WO2003024392 (Claim 2; FIG. 93); WO200166689 (Example 6);Cross-references: LocusID:54894; NP_060233.2; NM_017763_1.

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein, Genbank accession no. AF455138) Lab. Invest. 82(11):1573-1582 (2002)); WO2003087306; US2003064397 (Claim 1; FIG. 1);WO200272596 (Claim 13; Page 54-55); WO200172962 (Claim 1; FIG. 4B);WO2003104270 (Claim 11); WO2003104270 (Claim 16); US2004005598 (Claim22); WO2003042661 (Claim 12); US2003060612 (Claim 12; FIG. 10);WO200226822 (Claim 23; FIG. 2); WO200216429 (Claim 12; FIG. 10);Cross-references: GI:22655488; AAN04080.1; AF455138_1.

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation channel, subfamily M, member 4, Genbank accession no.NM_017636) Xu, X. Z., et al. Proc. Natl. Acad. Sci. U.S.A. 98(19):10692-10697 (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278(33):30813-30820 (2003)); US2003143557 (Claim 4); WO200040614 (Claim 14;Page 100-103); WO200210382 (Claim 1; FIG. 9A); WO2003042661 (Claim 12);WO200230268 (Claim 27; Page 391); US2003219806 (Claim 4); WO200162794(Claim 14; FIG. 1A-D); Cross-references: MIM:606936; NP_060106.2;NM_017636_1.

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor, Genbank accession no. NP_003203 or NM_003212)Ciccodicola, A., et al. EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.Genet. 49 (3):555-565 (1991)); US2003224411 (Claim 1); WO2003083041(Example 1); WO2003034984 (Claim 12); WO200288170 (Claim 2; Page 52-53);WO2003024392 (Claim 2; FIG. 58); WO200216413 (Claim 1; Page 94-95, 105);WO200222808 (Claim 2; FIG. 1); U.S. Pat. No. 5,854,399 (Example 2; Col17-18); U.S. Pat. No. 5,792,616 (FIG. 2); Cross-references: MIM:187395;NP_003203.1; NM_003212_1.

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792 Genbank accession no. M26004) Fujisaku et al.(1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J., et al. J. Exp.Med. 167, 1047-1066, 1988; Moore M., et al. Proc. Natl. Acad. Sci.U.S.A. 84, 9194-9198, 1987; Barel M., et al. Mol. Immunol. 35,1025-1031, 1998; Weis J. J., et al. Proc. Natl. Acad. Sci. U.S.A. 83,5639-5643, 1986; Sinha S. K., et al. (1993) J. Immunol. 150, 5311-5320;WO2004045520 (Example 4); US2004005538 (Example 1); WO2003062401 (Claim9); WO2004045520 (Example 4); WO9102536 (FIGS. 9.1-9.9); WO2004020595(Claim 1); Accession: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79β, CD790, IGb (immunoglobulin-associated beta), B29,Genbank accession no. NM_000626 or 11038674) Proc. Natl. Acad. Sci.U.S.A. (2003) 100 (7):4126-4131, Blood (2002) 100 (9):3068-3076, Mulleret al. (1992) Eur. J. Immunol. 22 (6):1621-1625); WO2004016225 (claim 2,FIG. 140); WO2003087768, US2004101874 (claim 1, page 102); WO2003062401(claim 9); WO200278524 (Example 2); US2002150573 (claim 5, page 15);U.S. Pat. No. 5,644,033; WO2003048202 (claim 1, pages 306 and 309); WO99/558658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B); WO200055351(claim 11, pages 1145-1146); Cross-references: MIM:147245; NP_000617.1;NM_000626_1.

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C, Genbank accession no. NM_030764,AY358130) Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54(2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad. Sci.U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al. (2001) Biochem.Biophys. Res. Commun. 280 (3):768-775; WO2004016225 (Claim 2);WO2003077836; WO200138490 (Claim 5; FIG. 18D-1-18D-2); WO2003097803(Claim 12); WO2003089624 (Claim 25); Cross-references: MIM:606509;NP_110391.2; NM_030764_1.

(17) HER2 (ErbB2, Genbank accession no. M11730) Coussens L., et al.Science (1985) 230(4730):1132-1139); Yamamoto T., et al. Nature 319,230-234, 1986; Semba K., et al. Proc. Natl. Acad. Sci. U.S.A. 82,6497-6501, 1985; Swiercz J. M., et al. J. Cell Biol. 165, 869-880, 2004;Kuhns J. J., et al. J. Biol. Chem. 274, 36422-36427, 1999; Cho H.-S., etal. Nature 421, 756-760, 2003; Ehsani A., et al. (1993) Genomics 15,426-429; WO2004048938 (Example 2); WO2004027049 (FIG. 1I); WO2004009622;WO2003081210; WO2003089904 (Claim 9); WO2003016475 (Claim 1);US2003118592; WO2003008537 (Claim 1); WO2003055439 (Claim 29; FIG.1A-B); WO2003025228 (Claim 37; FIG. 5C); WO200222636 (Example 13; Page95-107); WO200212341 (Claim 68; FIG. 7); WO200213847 (Page 71-74);WO200214503 (Page 114-117); WO200153463 (Claim 2; Page 41-46);WO200141787 (Page 15); WO200044899 (Claim 52; FIG. 7); WO200020579(Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3; Col 31-38);WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7); WO2004043361(Claim 7); WO2004022709; WO200100244 (Example 3; FIG. 4); Accession:P04626; EMBL; M11767; AAA35808.1. EMBL; M11761; AAA35808.1.

(18) NCA (CEACAM6, Genbank accession no. M18728); Barnett T., et al.Genomics 3, 59-66, 1988; Tawaragi Y., et al. Biochem. Biophys. Res.Commun. 150, 89-96, 1988; Strausberg R. L., et al. Proc. Natl. Acad.Sci. U.S.A. 99:16899-16903, 2002; WO2004063709; EP1439393 (Claim 7);WO2004044178 (Example 4); WO2004031238; WO2003042661 (Claim 12);WO200278524 (Example 2); WO200286443 (Claim 27; Page 427); WO200260317(Claim 2); Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. EMBL;M18728.

(19) MDP (DPEP1, Genbank accession no. BC017023) Proc. Natl. Acad. Sci.U.S.A. 99 (26):16899-16903 (2002)); WO2003016475 (Claim 1); WO200264798(Claim 33; Page 85-87); JP05003790 (FIG. 6-8); WO9946284 (FIG. 9);Cross-references: MIM:179780; AAH17023.1; BC017023_1.

(20) IL20Rα (IL20Ra, ZCYTOR7, Genbank accession no. AF184971); Clark H.F., et al. Genome Res. 13, 2265-2270, 2003; Mungall A. J., et al. Nature425, 805-811, 2003; Blumberg H., et al. Cell 104, 9-19, 2001; DumoutierL., et al. J. Immunol. 167, 3545-3549, 2001; Parrish-Novak J., et al. J.Biol. Chem. 277, 47517-47523, 2002; Pletnev S., et al. (2003)Biochemistry 42:12617-12624; Sheikh F., et al. (2004) J. Immunol. 172,2006-2010; EP1394274 (Example 11); US2004005320 (Example 5);WO2003029262 (Page 74-75); WO2003002717 (Claim 2; Page 63); WO200222153(Page 45-47); US2002042366 (Page 20-21); WO200146261 (Page 57-59);WO200146232 (Page 63-65); WO9837193 (Claim 1; Page 55-59); Accession:Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession no. AF229053) Gary S. C.,et al. Gene 256, 139-147, 2000; Clark H. F., et al. Genome Res. 13,2265-2270, 2003; Strausberg R. L., et al. Proc. Natl. Acad. Sci. U.S.A.99, 16899-16903, 2002; US2003186372 (Claim 11); US2003186373 (Claim 11);US2003119131 (Claim 1; FIG. 52); US2003119122 (Claim 1; FIG. 52);US2003119126 (Claim 1); US2003119121 (Claim 1; FIG. 52); US2003119129(Claim 1); US2003119130 (Claim 1); US2003119128 (Claim 1; FIG. 52);US2003119125 (Claim 1); WO2003016475 (Claim 1); WO200202634 (Claim 1).

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession no.NM_004442) Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991)Oncogene 10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998),Int. Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12);WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583(Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page 42);Cross-references: MIM:600997; NP_004433.2; NM_004442_1.

(23) ASLG659 (B7h, Genbank accession no. AX092328) US20040101899 (Claim2); WO2003104399 (Claim 11); WO2004000221 (FIG. 3); US2003165504 (Claim1); US2003124140 (Example 2); US2003065143 (FIG. 60); WO2002102235(Claim 13; Page 299); US2003091580 (Example 2); WO200210187 (Claim 6;FIG. 10); WO200194641 (Claim 12; FIG. 7b); WO200202624 (Claim 13; FIG.1A-1B); US2002034749 (Claim 54; Page 45-46); WO200206317 (Example 2;Page 320-321, Claim 34; Page 321-322); WO200271928 (Page 468-469);WO200202587 (Example 1; FIG. 1); WO200140269 (Example 3; Pages 190-192);WO200036107 (Example 2; Page 205-207); WO2004053079 (Claim 12);WO2003004989 (Claim 1); WO200271928 (Page 233-234, 452-453); WO 0116318.

(24) PSCA (Prostate stem cell antigen precursor, Genbank accession no.AJ297436) Reiter R. E., et al. Proc. Natl. Acad. Sci. U.S.A. 95,1735-1740, 1998; Gu Z., et al. Oncogene 19, 1288-1296, 2000; Biochem.Biophys. Res. Commun. (2000) 275(3):783-788; WO2004022709; EP1394274(Example 11); US2004018553 (Claim 17); WO2003008537 (Claim 1);WO200281646 (Claim 1; Page 164); WO2003003906 (Claim 10; Page 288);WO200140309 (Example 1; FIG. 17); US2001055751 (Example 1; FIG. 1b);WO200032752 (Claim 18; FIG. 1); WO9851805 (Claim 17; Page 97); WO9851824(Claim 10; Page 94); WO9840403 (Claim 2; FIG. 1B); Accession: O43653;EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession No. AY260763); AAP14954 lipoma HMGICfusion-partner-like protein/pid=AAP14954.1—Homo sapiens Species: Homosapiens (human) WO2003054152 (Claim 20); WO2003000842 (Claim 1);WO2003023013 (Example 3, Claim 20); US2003194704 (Claim 45);Cross-references: GI:30102449; AAP14954.1; AY260763_1.

(26) BAFF-R (B cell—activating factor receptor, BLyS receptor 3, BR3,Genbank accession No. AF116456); BAFF receptor/pid=NP_443177.1—Homosapiens Thompson, J. S., et al. Science 293 (5537), 2108-2111 (2001);WO2004058309; WO2004011611; WO2003045422 (Example; Page 32-33);WO2003014294 (Claim 35; FIG. 6B); WO2003035846 (Claim 70; Page 615-616);WO200294852 (Col 136-137); WO200238766 (Claim 3; Page 133); WO200224909(Example 3; FIG. 3); Cross-references: MIM:606269; NP_443177.1;NM_052945_1; AF132600.

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814, Genbank accession No. AK026467); Wilson et al.(1991) J. Exp. Med. 173:137-146; WO2003072036 (Claim 1; FIG. 1);Cross-references: MIM:107266; NP_001762.1; NM_001771_1.

(28) CD79a (CD79A, CD79α, immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation), pI: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2, Genbank accession No. NP_001774.10) WO2003088808,US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4, pages13-14); WO9958658 (claim 13, FIG. 16); WO9207574 (FIG. 1); U.S. Pat. No.5,644,033; Ha et al. (1992) J. Immunol. 148(5):1526-1531; Mueller et al.(1992) Eur. J. Biochem. 22:1621-1625; Hashimoto et al. (1994)Immunogenetics 40(4):287-295; Preud'homme et al. (1992) Clin. Exp.Immunol. 90(1):141-146; Yu et al. (1992) J. Immunol. 148(2) 633-637;Sakaguchi et al. (1988) EMBO J. 7(11):3457-3464.

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa,pI: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3, Genbank accessionNo. NP_001707.1) WO2004040000; WO2004015426; US2003105292 (Example 2);U.S. Pat. No. 6,555,339 (Example 2); WO200261087 (FIG. 1); WO200157188(Claim 20, page 269); WO200172830 (pages 12-13); WO200022129 (Example 1,pages 152-153, Example 2, pages 254-256); WO9928468 (claim 1, page 38);U.S. Pat. No. 5,440,021 (Example 2, col 49-52); WO9428931 (pages 56-58);WO9217497 (claim 7, FIG. 5); Dobner et al. (1992) Eur. J. Immunol.22:2795-2799; Barella et al. (1995) Biochem. J. 309:773-779.

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+ T lymphocytes); 273 aa, pI:6.56 MW: 30820 TM: 1 [P] Gene Chromosome: 6p21.3, Genbank accession No.NP_002111.1) Tonnelle et al. (1985) EMBO J. 4(11):2839-2847; Jonsson etal. (1989) Immunogenetics 29(6):411-413; Beck et al. (1992) J. Mol.Biol. 228:433-441; Strausberg et al. (2002) Proc. Natl. Acad. Sci USA99:16899-16903; Servenius et al. (1987) J. Biol. Chem. 262:8759-8766;Beck et al. (1996) J. Mol. Biol. 255:1-13; Naruse et al. (2002) TissueAntigens 59:512-519; WO9958658 (claim 13, FIG. 15); U.S. Pat. No.6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col 168-170); U.S. Pat.No. 6,011,146 (col 145-146); Kasahara et al. (1989) Immunogenetics30(1):66-68; Larhammar et al. (1985) J. Biol. Chem. 260(26):14111-14119.

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pI: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3, Genbank accession No.NP_002552.2) Le et al. (1997) FEBS Lett. 418(1-2):195-199; WO2004047749;WO2003072035 (claim 10); Touchman et al. (2000) Genome Res. 10:165-173;WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1);WO2003029277 (page 82).

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCEFull maeaity . . . tafrfpd (1.359; 359 aa), pI: 8.66, MW: 40225 TM: 1[P] Gene Chromosome: 9p13.3, Genbank accession No. NP_001773.1)WO2004042346 (claim 65); WO2003026493 (pages 51-52, 57-58); WO200075655(pages 105-106); Von Hoegen et al. (1990) J. Immunol. 144(12):4870-4877;Strausberg et al. (2002) Proc. Natl. Acad. Sci USA 99:16899-16903.

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pI:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12, Genbank accession No.NP_005573.1) US2002193567; WO9707198 (claim 11, pages 39-42); Miura etal. (1996) Genomics 38(3):299-304; Miura et al. (1998) Blood92:2815-2822; WO2003083047; WO9744452 (claim 8, pages 57-61);WO200012130 (pages 24-26).

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa, pI: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22, Genbank accession No.NP_443170.1) WO2003077836; WO200138490 (claim 6, FIG. 18E-1-18-E-2);Davis et al. (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777;WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7).

(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); 977 aa, pI: 6.88 MW: 106468 TM: 1 [P] GeneChromosome: 1q21, Genbank accession No. Human:AF343662, AF343663,AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187,AY358085; Mouse:AK089756, AY158090, AY506558; NP_112571.1. WO2003024392(claim 2, FIG. 97); Nakayama et al. (2000) Biochem. Biophys. Res.Commun. 277(1):124-127; WO2003077836; WO200138490 (claim 3, FIG.18B-1-18B-2).

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa, NCBI Accession: AAD55776, AAF91397, AAG49451, NCBIRefSeq: NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;Genbank accession No. AF179274; AY358907, CAF85723, CQ782436WO2004074320; JP2004113151; WO2003042661; WO2003009814; EP1295944 (pages69-70); WO200230268 (page 329); WO200190304; US2004249130; US2004022727;WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579;Horie et al. (2000) Genomics 67:146-152; Uchida et al. (1999) Biochem.Biophys. Res. Commun. 266:593-602; Liang et al. (2000) Cancer Res.60:4907-12; Glynne-Jones et al. (2001) Int J Cancer. October 15;94(2):178-84.

(37) PMEL17 (silver homolog; SILV; D12S53E; PMEL17; SI; SIL); ME20;gp100) BC001414; BT007202; M32295; M77348; NM_006928; McGlinchey, R. P.et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106 (33), 13731-13736;Kummer, M. P. et al. (2009) J. Biol. Chem. 284 (4), 2296-2306.

(38) TMEFF1 (transmembrane protein with EGF-like and twofollistatin-like domains 1; Tomoregulin-1); H7365; C9orf2; C9ORF2;U19878; X83961; NM_080655; NM_003692; Harms, P. W. (2003) Genes Dev. 17(21), 2624-2629; Gery, S. et al. (2003) Oncogene 22 (18):2723-2727.

(39) GDNF-Ra1 (GDNF family receptor alpha 1; GFRA1; GDNFR; GDNFRA;RETL1; TRNR1; RET1L; GDNFR-alpha1; GFR-ALPHA-1); U95847; BC014962;NM_145793 NM_005264; Kim, M. H. et al. (2009) Mol. Cell. Biol. 29 (8),2264-2277; Treanor, J. J. et al. (1996) Nature 382 (6586):80-83.

(40) Ly6E (lymphocyte antigen 6 complex, locus E; Ly67, RIG-E,SCA-2,TSA-1); NP_002337.1; NM_002346.2; de Nooij-van Dalen, A. G. et al.(2003) Int. J. Cancer 103 (6), 768-774; Zammit, D. J. et al. (2002) Mol.Cell. Biol. 22 (3):946-952.

(41) TMEM46 (shisa homolog 2 (Xenopus laevis); SHISA2); NP_001007539.1;NM_001007538.1; Furushima, K. et al. (2007) Dev. Biol. 306 (2), 480-492;Clark, H. F. et al. (2003) Genome Res. 13 (10):2265-2270.

(42) Ly6G6D (lyrnphocyte antigen 6 complex, locus G6D; Ly6-D, MEGT1);NP_067079.2; NM_021246.2; Mallya, M. et al. (2002) Genomics 80(1):113-123; Ribas, G. et al. (1999) J. Immunol. 163 (1):278-287.

(43) LGR5 (leucine-rich repeat-containing G protein-coupled receptor 5;GPR49, GPR67); NP_003658.1; NM_003667.2; Salanti, G. et al. (2009) Am.J. Epidemiol. 170 (5):537-545; Yamamoto, Y. et al. (2003) Hepatology 37(3):528-533.

(44) RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12;Hs.168114; RET51; RET-ELE1); NP_066124.1; NM_020975.4; Tsukamoto, H. etal. (2009) Cancer Sci. 100 (10):1895-1901; Narita, N. et al. (2009)Oncogene 28 (34):3058-3068.

(45) LY6K (lymphocyte antigen 6 complex, locus K; LY6K; HSJ001348;FLJ35226); NP_059997.3; NM_017527.3; Ishikawa, N. et al. (2007) CancerRes. 67 (24):11601-11611; de Nooij-van Dalen, A. G. et al. (2003) Int.J. Cancer 103 (6):768-774.

(46) GPR19 (G protein-coupled receptor 19; Mm.4787); NP_006134.1;NM_006143.2; Montpetit, A. and Sinnett, D. (1999) Hum. Genet. 105(1-2):162-164; O'Dowd, B. F. et al. (1996) FEBS Lett. 394 (3):325-329.

(47) GPR54 (KISS1 receptor; KISS1R; GPR54; HOT7T175; AXOR12);NP_115940.2; NM_032551.4; Navenot, J. M. et al. (2009) Mol. Pharmacol.75 (6):1300-1306; Hata, K. et al. (2009) Anticancer Res. 29 (2):617-623.

(48) ASPHD1 (aspartate beta-hydroxylase domain containing 1; LOC253982);NP_859069.2; NM_181718.3; Gerhard, D. S. et al. (2004) Genome Res. 14(10B):2121-2127.

(49) Tyrosinase (TYR; OCAIA; OCA1A; tyrosinase; SHEP3); NP_000363.1;NM_000372.4; Bishop, D. T. et al. (2009) Nat. Genet. 41 (8):920-925;Nan, H. et al. (2009) Int. J. Cancer 125 (4):909-917.

(50) TMEM118 (ring finger protein, transmembrane 2; RNFT2; FLJ14627);NP_001103373.1; NM_001109903.1; Clark, H. F. et al. (2003) Genome Res.13 (10):2265-2270; Scherer, S. E. et al. (2006) Nature 440(7082):346-351.

(51) GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e); NP_078807.1; NM_024531.3; Ericsson, T. A. et al. (2003)Proc. Natl. Acad. Sci. U.S.A. 100 (11):6759-6764; Takeda, S. et al.(2002) FEBS Lett. 520 (1-3):97-101.

(52) CD33, a member of the sialic acid binding, immunoglobulin-likelectin family, is a 67-kDa glycosylated transmembrane protein. CD33 isexpressed on most myeloid and monocytic leukemia cells in addition tocommitted myelomonocytic and erythroid progenitor cells. It is not seenon the earliest pluripotent stem cells, mature granulocytes, lymphoidcells, or nonhematopoietic cells (Sabbath et al., (1985) J. Clin.Invest. 75:756-56; Andrews et al., (1986) Blood 68:1030-5). CD33contains two tyrosine residues on its cytoplasmic tail, each of which isfollowed by hydrophobic residues similar to the immunoreceptortyrosine-based inhibitory motif (ITIM) seen in many inhibitoryreceptors.

(53) CLL-1 (CLEC12A, MICL, and DCAL2), encodes a member of the C-typelectin/C-type lectin-like domain (CTL/CTLD) superfamily. Members of thisfamily share a common protein fold and have diverse functions, such ascell adhesion, cell-cell signaling, glycoprotein turnover, and roles ininflammation and immune response. The protein encoded by this gene is anegative regulator of granulocyte and monocyte function. Severalalternatively spliced transcript variants of this gene have beendescribed, but the full-length nature of some of these variants has notbeen determined. This gene is closely linked to other CTL/CTLDsuperfamily members in the natural killer gene complex region onchromosome 12p13 (Drickamer K (1999) Curr. Opin. Struct. Biol. 9(5):585-90; van Rhenen A, et al., (2007) Blood 110 (7):2659-66; Chen CH, et al. (2006) Blood 107 (4):1459-67; Marshall A S, et al. (2006) Eur.J. Immunol. 36 (8):2159-69; Bakker A B, et al. (2005) Cancer Res. 64(22):8443-50; Marshall A S, et al. (2004) J. Biol. Chem. 279(15):14792-802). CLL-1 has been shown to be a type II transmembranereceptor comprising a single C-type lectin-like domain (which is notpredicted to bind either calcium or sugar), a stalk region, atransmembrane domain and a short cytoplasmic tail containing an ITIMmotif.

In any of the antibody embodiments of the disclosure, an antibody ishumanized. In one embodiment, an antibody comprises HVRs as in any ofthe embodiments of the disclosure, and further comprises a humanacceptor framework, e.g. a human immunoglobulin framework or a humanconsensus framework. In certain embodiments, the human acceptorframework is the human VL kappa I consensus (VLKI) framework and/or theVH framework VH1. In certain embodiments, the human acceptor frameworkis the human VL kappa I consensus (VLKI) framework and/or the VHframework VH1 comprising any one of the following mutations.

In another embodiment, the antibody comprises a VH as in any of theembodiments provided herein, and a VL as in any of the embodimentsprovided herein.

In a further embodiment of the disclosure, an antibody according to anyof the embodiments herein is a monoclonal antibody, including a humanantibody. In one embodiment, an antibody is an antibody fragment, e.g.,a Fv, Fab, Fab′, scFv, diabody, or F(ab′)2 fragment. In anotherembodiment, the antibody is a substantially full length antibody, e.g.,an IgG1 antibody, IgG2a antibody or other antibody class or isotype asdefined herein. In some particular embodiments, the antibody is selectedfrom anti-HER2, anti-CD22, anti-CD33, anti-Napi2b, and anti-CLL-1.

In a further embodiment, an antibody according to any of the embodimentsherein may incorporate any of the features, singly or in combination, asdescribed herein.

B. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤1 μM, ≤100 nM, ≤50 nM, ≤10 nM, ≤5 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM, and optionally is ≥10⁻¹³ M. (e.g. 10⁻⁸ M orless, e.g. from 10⁻⁸ M to 10⁻¹³ M, e.g., from 10⁻⁹ M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 l/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACORE®-2000, BIACORE®-T200 or a BIACORE®-3000(BIAcore, Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CM5chips at ˜10 response units (RU). Briefly, carboxymethylated dextranbiosensor chips (CM5, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) and/or HBS-P (0.01 M Hepes pH7.4, 0.15M NaCl, 0.005% Surfactant P20)before injection at a flow rate of 5 μl/minute and/or 30 μl/minute toachieve approximately 10 response units (RU) of coupled protein.Following the injection of antigen, 1 M ethanolamine is injected toblock unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate ofapproximately 25 μl/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIACORE® Evaluation Software version 3.2) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (Kd) is calculated as the ratiok_(off)/k_(on). See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999). If the on-rate exceeds 10⁶ M⁻¹ s⁻¹ by the surface plasmonresonance assay describe herein, then the on-rate can be determined byusing a fluorescent quenching technique that measures the increase ordecrease in fluorescence emission intensity (excitation=295 nm;emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-seriesSLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette.

C. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)2, Fv, and scFv fragments, and other fragments describedherein. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)2 fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

D. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

E. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3): 185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described herein.

F. Library-Derived Antibodies

Antibodies of the disclosure may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self- and also self-antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

G. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. The term “multispecific antibody”is used in the broadest sense and specifically covers an antibodycomprising an antigen-binding domain that has polyepitopic specificity(i.e., is capable of specifically binding to two, or more, differentepitopes on one biological molecule or is capable of specificallybinding to epitopes on two, or more, different biological molecules). Insome embodiments, multispecific antibodies are monoclonal antibodiesthat have binding specificities for at least two different sites. Insome embodiments, an antigen-binding domain of a multispecific antibody(such as a bispecific antibody) comprises two VH/VL units, wherein afirst VH/VL unit specifically binds to a first epitope and a secondVH/VL unit specifically binds to a second epitope, wherein each VH/VLunit comprises a heavy chain variable domain (VH) and a light chainvariable domain (VL). Such multispecific antibodies include, but are notlimited to, full length antibodies, antibodies having two or more VL andVH domains, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies,bispecific diabodies and triabodies, antibody fragments that have beenlinked covalently or non-covalently. A VH/VL unit that further comprisesat least a portion of a heavy chain variable region and/or at least aportion of a light chain variable region may also be referred to as an“arm” or “hemimer” or “half antibody.” In some embodiments, a hemimercomprises a sufficient portion of a heavy chain variable region to allowintramolecular disulfide bonds to be formed with a second hemimer. Insome embodiments, a hemimer comprises a knob mutation or a holemutation, for example, to allow heterodimerization with a second hemimeror half antibody that comprises a complementary hole mutation or knobmutation. Knob mutations and hole mutations are discussed furtherherein.

In certain embodiments, a multispecific antibody provided herein may bea bispecific antibody. The term “bispecific antibody” is used in thebroadest sense and covers a multispecific antibody comprising anantigen-binding domain that is capable of specifically binding to twodifferent epitopes on one biological molecule or is capable ofspecifically binding to epitopes on two different biological molecules.A bispecific antibody may also be referred to herein as having “dualspecificity” or as being “dual specific.” Bispecific antibodies can beprepared as full length antibodies or antibody fragments. The term“biparatopic antibody” as used herein, refers to a bispecific antibodywhere a first antigen-binding domain and a second antigen-binding domainbind to two different epitopes on the same antigen molecule or it maybind to epitopes on two different antigen molecules.

In some embodiments, the first antigen-binding domain and the secondantigen-binding domain of the biparatopic antibody may bind the twoepitopes within one and the same antigen molecule (intramolecularbinding). For example, the first antigen-binding domain and the secondantigen-binding domain of the biparatopic antibody may bind to twodifferent epitopes on the same antibody molecule. In certainembodiments, the two different epitopes that a biparatopic antibodybinds are epitopes that are not normally bound at the same time by onemonospecific antibody, such as e.g. a conventional antibody or oneimmunoglobulin single variable domain.

In some embodiments, the first antigen-binding domain and the secondantigen-binding domain of the biparatopic antibody may bind epitopeslocated within two distinct antigen molecules.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168), WO2009/089004, US2009/0182127, US2011/0287009,Marvin and Zhu, Acta Pharmacol. Sin. (2005) 26(6):649-658, andKontermann (2005) Acta Pharmacol. Sin., 26:1-9). The term“knob-into-hole” or “KnH” technology as used herein refers to thetechnology directing the pairing of two polypeptides together in vitroor in vivo by introducing a protuberance (knob) into one polypeptide anda cavity (hole) into the other polypeptide at an interface in which theyinteract. For example, KnHs have been introduced in the Fc:Fc bindinginterfaces, CL:CH1 interfaces or VH/VL interfaces of antibodies (see,e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, andZhu et al., 1997, Protein Science 6:781-788). In some embodiments, KnHsdrive the pairing of two different heavy chains together during themanufacture of multispecific antibodies. For example, multispecificantibodies having KnH in their Fc regions can further comprise singlevariable domains linked to each Fc region, or further comprise differentheavy chain variable domains that pair with similar or different lightchain variable domains. KnH technology can be also be used to pair twodifferent receptor extracellular domains together or any otherpolypeptide sequences that comprises different target recognitionsequences (e.g., including affibodies, peptibodies and other Fcfusions).

The term “knob mutation” as used herein refers to a mutation thatintroduces a protuberance (knob) into a polypeptide at an interface inwhich the polypeptide interacts with another polypeptide. In someembodiments, the other polypeptide has a hole mutation.

A “protuberance” refers to at least one amino acid side chain whichprojects from the interface of a first polypeptide and is thereforepositionable in a compensatory cavity in the adjacent interface (i.e.the interface of a second polypeptide) so as to stabilize theheteromultimer, and thereby favor heteromultimer formation overhomomultimer formation, for example. The protuberance may exist in theoriginal interface or may be introduced synthetically (e.g. by alteringnucleic acid encoding the interface). In some embodiments, nucleic acidencoding the interface of the first polypeptide is altered to encode theprotuberance. To achieve this, the nucleic acid encoding at least one“original” amino acid residue in the interface of the first polypeptideis replaced with nucleic acid encoding at least one “import” amino acidresidue which has a larger side chain volume than the original aminoacid residue. It will be appreciated that there can be more than oneoriginal and corresponding import residue. The side chain volumes of thevarious amino residues are shown, for example, in Table 1 ofUS2011/0287009. A mutation to introduce a “protuberance” may be referredto as a “knob mutation.”

In some embodiments, import residues for the formation of a protuberanceare naturally occurring amino acid residues selected from arginine (R),phenylalanine (F), tyrosine (Y) and tryptophan (W). In some embodiments,an import residue is tryptophan or tyrosine. In some embodiment, theoriginal residue for the formation of the protuberance has a small sidechain volume, such as alanine, asparagine, aspartic acid, glycine,serine, threonine or valine.

A “cavity” refers to at least one amino acid side chain which isrecessed from the interface of a second polypeptide and thereforeaccommodates a corresponding protuberance on the adjacent interface of afirst polypeptide. The cavity may exist in the original interface or maybe introduced synthetically (e.g. by altering nucleic acid encoding theinterface). In some embodiments, nucleic acid encoding the interface ofthe second polypeptide is altered to encode the cavity. To achieve this,the nucleic acid encoding at least one “original” amino acid residue inthe interface of the second polypeptide is replaced with DNA encoding atleast one “import” amino acid residue which has a smaller side chainvolume than the original amino acid residue. It will be appreciated thatthere can be more than one original and corresponding import residue. Insome embodiments, import residues for the formation of a cavity arenaturally occurring amino acid residues selected from alanine (A),serine (S), threonine (T) and valine (V). In some embodiments, an importresidue is serine, alanine or threonine. In some embodiments, theoriginal residue for the formation of the cavity has a large side chainvolume, such as tyrosine, arginine, phenylalanine or tryptophan. Amutation to introduce a “cavity” may be referred to as a “holemutation.”

The protuberance is “positionable” in the cavity which means that thespatial location of the protuberance and cavity on the interface of afirst polypeptide and second polypeptide respectively and the sizes ofthe protuberance and cavity are such that the protuberance can belocated in the cavity without significantly perturbing the normalassociation of the first and second polypeptides at the interface. Sinceprotuberances such as Tyr, Phe and Trp do not typically extendperpendicularly from the axis of the interface and have preferredconformations, the alignment of a protuberance with a correspondingcavity may, in some instances, rely on modeling the protuberance/cavitypair based upon a three-dimensional structure such as that obtained byX-ray crystallography or nuclear magnetic resonance (NMR). This can beachieved using widely accepted techniques in the art.

In some embodiments, a knob mutation in an IgG1 constant region isT366W. In some embodiments, a hole mutation in an IgG1 constant regioncomprises one or more mutations selected from T366S, L368A and Y407V. Insome embodiments, a hole mutation in an IgG1 constant region comprisesT366S, L368A and Y407V.

In some embodiments, a knob mutation in an IgG4 constant region isT366W. In some embodiments, a hole mutation in an IgG4 constant regioncomprises one or more mutations selected from T366S, L368A, and Y407V.In some embodiments, a hole mutation in an IgG4 constant regioncomprises T366S, L368A, and Y407V.

Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229: 81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (Hollingeret al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and usingsingle-chain Fv (sFv) dimers (Gruber et al., J. Immunol., 152:5368(1994)); and preparing trispecific antibodies as described, e.g., inTutt et al. J. Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies” or “dual-variable domainimmunoglobulins” (DVDs) are also included herein (US 2006/0025576A1, andWu et al. (2007) Nature Biotechnology).

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the disclosure contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half-lifedeterminations can also be performed using methods known in the art(Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056 which is incorporated byreference in its entirety). Such Fc mutants include Fc mutants withsubstitutions at two or more of amino acid positions 265, 269, 270, 297and 327, including the so-called “DANA” Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581 which isincorporated by reference in its entirety).

In certain embodiments Pro329 of a wild-type human Fc region issubstituted with glycine or arginine or an amino acid residue largeenough to destroy the proline sandwich within the Fc/Fcγ receptorinterface, that is formed between the proline329 of the Fc andtryptophan residues Trp 87 and Trp 110 of FcgRIII (Sondermann et al.:Nature 406, 267-273 (20 Jul. 2000)). In a further embodiment, at leastone further amino acid substitution in the Fc variant is S228P, E233P,L234A, L235A, L235E, N297A, N297D, or P331S and still in anotherembodiment said at least one further amino acid substitution is L234Aand L235A of the human IgG1 Fc region or S228P and L235E of the humanIgG4 Fc region (U.S. Pat. No. 8,969,526 which is incorporated byreference in its entirety).

In certain embodiments, a polypeptide comprises the Fc variant of awild-type human IgG Fc region wherein the polypeptide has Pro329 of thehuman IgG Fc region substituted with glycine and wherein the Fc variantcomprises at least two further amino acid substitutions at L234A andL235A of the human IgG1 Fc region or S228P and L235E of the human IgG4Fc region, and wherein the residues are numbered according to the EUindex of Kabat (U.S. Pat. No. 8,969,526 which is incorporated byreference in its entirety). In certain embodiments, the polypeptidecomprising the P329G, L234A and L235A substitutions exhibit a reducedaffinity to the human FcγRIIIA and FcγRIIA, for down-modulation of ADCCto at least 20% of the ADCC induced by the polypeptide comprising thewild type human IgG Fc region, and/or for down-modulation of ADCP (U.S.Pat. No. 8,969,526 which is incorporated by reference in its entirety).

In a specific embodiment the polypeptide comprising an Fc variant of awild type human Fc polypeptide comprises a triple mutation: an aminoacid substitution at position Pro329, a L234A and a L235A mutation(P329/LALA) (U.S. Pat. No. 8,969,526 which is incorporated by referencein its entirety). In specific embodiments, the polypeptide comprises thefollowing amino acid substitutions: P329G, L234A, and L235A.

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half-lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. Nos.5,648,260; 5,624,821; and WO 94/29351 concerning other examples of Fcregion variants.

H. Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “THIOMAB™ antibody,” in which one or moreresidues of an antibody are substituted with cysteine residues. Inparticular embodiments, the substituted residues occur at accessiblesites of the antibody. By substituting those residues with cysteine,reactive thiol groups are thereby positioned at accessible sites of theantibody and may be used to conjugate the antibody to the drug moiety tocreate an immunoconjugate, as described further herein. In certainembodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;K149 (Kabat numbering) of the light chain; A118 (EU numbering) of theheavy chain; and S400 (EU numbering) of the heavy chain Fc region.Cysteine engineered antibodies may be generated as described, e.g., inU.S. Pat. No. 7,521,541.

In some embodiments, a THIOMAB™ antibody comprises one of the heavy orlight chain cysteine substitutions listed in Table A below.

TABLE A Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # LC T 22 22 22 LC K 39 39 39 LC Y 49 49 49LC Y 55 55 55 LC T 85 85 85 LC T 97 97 97 LC I 106 106 106 LC R 108 108108 LC R 142 142 142 LC K 149 149 149 LC V 205 205 205 HC T 117 114 110HC A 143 140 136 HC L 177 174 170 HC L 182 179 175 HC T 190 187 183 HC T212 209 205 HC V 265 262 258 HC G 374 371 367 HC Y 376 373 369 HC E 385382 378 HC S 427 424 420 HC N 437 434 430 HC Q 441 438 434

In other embodiments, a THIOMAB™ antibody comprises one of the heavychain cysteine substitutions listed in Table B.

TABLE B Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # HC T 117 114 110 HC A 143 140 136 HC L 177174 170 HC L 182 179 175 HC T 190 187 183 HC T 212 209 205 HC V 265 262258 HC G 374 371 367 HC Y 376 373 369 HC E 385 382 378 HC S 427 424 420HC N 437 434 430 HC Q 441 438 434

In some other embodiments, a THIOMAB™ antibody comprises one of thelight chain cysteine substitutions listed in Table C.

TABLE C Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # LC I 106 106 106 LC R 108 108 108 LC R 142142 142 LC K 149 149 149

In some other embodiments, a THIOMAB™ antibody comprises one of theheavy or light chain cysteine substitutions listed in Table D.

TABLE D Screening GNE Kabat Chain Mutation Mutation Mutation (HC/LC)Residue Site # Site # Site # LC K 149 149 149 HC A 143 140 136 HC A 121118 114

Cysteine engineered antibodies which may be useful in the antibody-drugconjugates (ADC) of the disclosure in the treatment of cancer include,but are not limited to, antibodies against cell surface receptors andtumor-associated antigens (TAA). Tumor-associated antigens are known inthe art, and can be prepared for use in generating antibodies usingmethods and information which are well known in the art. In attempts todiscover effective cellular targets for cancer diagnosis and therapy,researchers have sought to identify transmembrane or otherwisetumor-associated polypeptides that are specifically expressed on thesurface of one or more particular type(s) of cancer cell as compared toon one or more normal non-cancerous cell(s). Often, suchtumor-associated polypeptides are more abundantly expressed on thesurface of the cancer cells as compared to on the surface of thenon-cancerous cells. The identification of such tumor-associated cellsurface antigen polypeptides has given rise to the ability tospecifically target cancer cells for destruction via antibody-basedtherapies.

Examples of tumor-associated antigens TAA include, but are not limitedto, TAA (1)-(53) listed herein. For convenience, information relating tothese antigens, all of which are known in the art, is listed herein andincludes names, alternative names, Genbank accession numbers and primaryreference(s), following nucleic acid and protein sequence identificationconventions of the National Center for Biotechnology Information (NCBI).Nucleic acid and protein sequences corresponding to TAA (1)-(53) areavailable in public databases such as GenBank. Tumor-associated antigenstargeted by antibodies include all amino acid sequence variants andisoforms possessing at least about 70%, 80%, 85%, 90%, or 95% sequenceidentity relative to the sequences identified in the cited references,or which exhibit substantially the same biological properties orcharacteristics as a TAA having a sequence found in the citedreferences. For example, a TAA having a variant sequence generally isable to bind specifically to an antibody that binds specifically to theTAA with the corresponding sequence listed. The sequences and disclosurein the reference specifically recited herein are expressly incorporatedby reference.

VI. Prodrug Preparation

Methods for the preparation of PBD monomer prodrugs and PBD dimerprodrugs within the scope of the present disclosure are describedelsewhere herein.

VII. Linker-Drug Conjugation

Conjugation of the linker to a PBD amine may suitably be done accordingto the methods of WO 2013/055987, WO 2015/023355 and WO 2015/095227,each of which is incorporated by reference herein in its entirety.

In some such embodiments, an activated linker as described elsewhereherein is combined with a solution of PBD monomer or dimer to form alinker-PBD conjugate. Generally, any solvent capable of providing asolution comprising from about 0.05 to about 1 mole per liter PBD issuitable. In some embodiments, the solvent is DCM. In some otherlinker-PBD conjugation embodiments, a solution of PBD, a stoichiometricexcess of triphosgene (or diphosgene or phosgene), and a base (e.g.,4-dimethylaminopyridine) is formed in a solvent (e.g., dry DCM). Thelinker intermediate having an alcohol moiety (as described elsewhereherein) is combined with the PBD solution to form a reaction mixturethat is stirred until the reaction is complete to form a product mixturecomprising the linker-PBD conjugate. The reaction mixture may suitablycomprise from about 0.005 moles per liter to about 0.5 moles per literof PBD, from about 2 to about 10 equivalents of linker intermediate perequivalent of PBD, and from about 0.02 to about 0.5 equivalents of base.After reaction completion, the linker-PBD conjugate may be isolated,such as by solvent evaporation, and purified by methods known in the artsuch as one or more of extraction, reverse phase high pressure liquidchromatography, ion exchange chromatography or flash chromatography.

VIII. Preparation of Disulfide Conjugate Compounds

In some embodiments, disulfide conjugate compounds of the disclosure offormula:

may be prepared by forming a reaction mixture comprising (1) a solventsystem comprising water, (2) a source of an antibody comprising at leastone cysteine having a sulfhydryl moiety, and (3) a stoichiometric excessof a source of a linker-PBD conjugate of the following formula:

The antibody, X_(L), R¹, R², Sp, n and D are as described elsewhereherein. The reaction mixture is reacted to form a mixture comprising thedisulfide conjugate compound of formula (II), wherein p is 1, 2, 3, 4,5, 6, 7 or 8. Although the individual disulfide conjugate compounds in amixture may have a p value of from 1 to 8, and some antibody moleculesin such a mixture may be unconjugated (p=0), the PBD to antibody ratiofor a plurality of formed disulfide conjugate compounds in the productmixture, expressed as the ratio of PBD equivalents to antibodyequivalents, is from about 1 to about 5, from about 1.5 to about 3, fromabout 1.5 to about 2.5, from about 1.7 to about 2.3, from about 1.8 toabout 2.2, or about 2.

In any of the various embodiments of such embodiments, the source of theantibody may be provided in solution in an aqueous buffer. In some suchembodiments, the buffer may suitably beN-(2-Acetamido)-aminoethanesulfonic acid (“ACES”); acetate salt;N-(2-Acetamido)-iminodiacetic acid (“ADA”); 2-Aminoethanesulfonic acid,Taurine (“AES”); 2-Amino-2-methyl-1-propanol (“AMP”);2-Amino-2-methyl-1,3-propanediol, Ammediol (“AMPD”);N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid(“AMPSO”); N,N-Bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (“BES”);N,N′-Bis(2-hydroxyethyl)-glycine (“Bicine”);[Bis-(2-hydroxyethyl)-imino]-tris-(hydroxymethylmethane) (“BIS-Tris”);1,3-Bis[tris(hydroxymethyl)-methylamino]propane (“BIS-Tris-Propane”);Dimethylarsinic acid (“Cacodylate”);3-(Cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (“CAPSO”);Cyclohexylaminoethanesulfonic acid (“CHES”); citric acid salt;3-[N-Bis(hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (“DIPSO”);N-(2-Hydroxyethyl)-piperazine-N′-ethanesulfonic acid (“HEPES”);N-(2-Hydroxyethyl)-piperazine-N′-3-propanesulfonic acid (“HEPPS, EPPS”);N-(2-Hydroxyethyl)-piperazine-N′-2-hydroxypropanesulfonic acid(“HEPPSO”); 2-(N-Morpholino)-ethanesulfonic acid (“MES”);3-(N-Morpholino)-propanesulfonic acid (“MOPS”);3-(N-Morpholino)-2-hydroxypropanesulfonic acid (“MOPSO”);Piperazine-N,N′-bis(2-ethanesulfonic acid) (“PIPES”);Piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (“POPSO”); salt ofsuccinic acid; 3-{[Tris(hydroxymethyl)-methyl]-amino}-propanesulfonicacid (“TAPS”); 3-[N-Tris(hydroxymethyl)-methylamino]-2-hydroxypropanesulfonic acid (“TAPSO”); 2-Aminoethanesulfonicacid, AES (“Taurine”); Triethanolamine (“TEA”);2-[Tris(hydroxymethyl)-methylamino]-ethanesulfonic acid (“TES”);N-[Tris(hydroxymethyl)-methyl]-glycine (“Tricine”);Tris(hydroxymethyl)-aminomethane (“TRIS”). In some such embodiments, thebuffer may be succinate or tris. The buffer concentration is suitablyabout 5 mM, about 10 mM, about 15 mM, about 20 mM, about 50 mM, about100 mM, or about 150 nM, such as from about 5 mM to about 150 mM, fromabout 5 mM to about 30 mM, from about 5 mM to about 20 mM, from about 5mM to about 10 mM, or from about 50 mM to about 100 nM. Theconcentration of the antibody in the buffer may be about 1 mg/mL, about5 mg/mL, about 10 mg/mL or more. The pH of the antibody solution issuitably from about 4 to about 8, from about 4 to about 6, or about 5.

In any of the various embodiments of such embodiments, the source of alinker-PBD conjugate compound (“activated linker-PBD conjugate”) maygenerally be formed by dissolving an activated hindered linker-PBDconjugate in a solvent comprising at least one polar aprotic solventselected from acetonitrile, tetrahydrofuran, ethyl acetate, acetone,N,N-dimethylformamide, dimethyl acetamide, dimethylsulfoxide, propyleneglycol, ethylene glycol and dichloromethane. In some embodiments, thesolvent comprises, predominantly comprises, or consists essentially ofN,N-dimethylformamide and/or dimethyl acetamide.

The source of the antibody and the source of the activated linker-PBDconjugate may suitably be admixed to form a reaction mixture. Theantibody concentration in the reaction mixture is suitably about 1mg/mL, about 5 mg/mL, about 10 mg/mL, about 15 mg/mL, about 20 mg/mL, orabout 25 mg/mL, and ranges thereof, such as from about 1 mg/mL to about25 mg/mL, from about 1 mg/mL to about 20 mg/mL, from about 1 mg/mL toabout 15 mg/mL, from about 1 mg/mL to about 10 mg/mL, from about 5 mg/mLto about 25 mg/mL, from about 5 mg/mL to about 20 mg/mL or from about 5mg/mL to about 15 mg/mL. The equivalent ratio of activated linker-PBDconjugate to the antibody is suitably about 2:1, about 3:1, about 5:1,about 10:1, about 15:1 or about 20:1, and ranges thereof, such as fromabout 2:1 to about 20:1, from about 3:1 to about 15:1, from about 3:1 toabout 10:1, or from about 5:1 to about 10:1. In some embodiments of thepresent disclosure, the reaction mixture solvent system predominantlycomprises water. In some other embodiments, the reaction mixture solventsystem may generally comprise at least 75 v/v % of a buffer as describedelsewhere herein and about 5 v/v %, about 10 v/v %, about 15 v/v %,about 20 v/v %, about 25 v/v % or about 30 v/v %, and ranges thereof,such as from about 5 v/v % to about 30 v/v %, from about 5 v/v % toabout 20 v/v %, from about 5 v/v % to about 15 v/v %, or about 10 v/v %of a polar aprotic solvent as described elsewhere herein. In some otherembodiments, the reaction mixture solvent system may generally compriseat least 50 v/v % of a buffer as described elsewhere herein and betweenabout 10 v/v % and about 50 v/v %, such as about 10 v/v %, about 20 v/v%, about 30 v/v % about 40 v/v % or above 50 v/v % propylene glycol orethylene glycol. Alternatively stated, the reaction mixture solventsystem may comprise, about 50 v/v %, about 60 v/v %, about 70 v/v %, 75v/v %, about 80 v/v %, about 85 v/v %, about 90 v/v % or about 95 v/v %water to about 95 v/v %, and ranges thereof, such as from about 50 v/v %to about 95 v/v % water, from about 75 v/v % to about 95 v/v % water,from about 80 v/v % to about 95 v/v % water or from about 85 v/v % toabout 95 v/v % water. The pH of the reaction mixture is suitably about5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0,about 8.5 or about 9.0, and ranges thereof, such as from about 5.0 toabout 9.0, from about 5.0 to about 9.0, from about 6.0 to about 9.0,from about 6.5 to about 9.0, or from about 7.0 to about 9.0, or fromabout 7.5 to about 8.5.

The reaction mixture is incubated at about 10° C., about 15° C., about20° C., about 25° C., about 30° C., about 35° C., about 40° C., about45° C. or about 50° C., and ranges thereof such as from about 10° C. toabout 50° C., from about 15° C. to about 45° C., from about 15° C. toabout 40° C., from about 20° C. to about 40° C., from about 20° C. toabout 35° C., or from about 20° C. to about 30° C. for about 0.5 hours,about 1 hour, about 4 hours, about 8 hours, about 12 hours, about 18hours about 24 hours, about 36 hours or about 48 hours, and rangesthereof, such as from about 0.5 hours to about 48 hours, or from about 1hour to about 24 hours to form a product mixture comprising thedisulfide conjugate compound of formula (II).

The product mixture may comprise at least 60 A % antibody-linker-PBDconjugate as determined by MS/LC, at least 65 area %, at least 70 area%, at least 75 area %, at least 80 area %, at least 85 area %, or atleast 90 area %. The product mixture may further comprise at least oneleaving group byproduct species. In some embodiments, the areapercentage of total leaving group byproduct species as compared to thearea percentage of formed disulfide conjugate compound as measured byMS/LC is less than 10 area %, less than 5 area %, less than 4 area %,less than 3 area %, less than 2 area %, less than 1 area %, less than0.5 area %. In the case of antibodies, and based on experimentalevidence to date, the leaving group byproduct species may comprise:

wherein X_(L) is as defined elsewhere herein and wherein Q refers to anX_(L) moiety not having a sulfur linking atom. Exemplary X andcorresponding Q are illustrated below:

The reaction mixture may further comprise unconjugated antibodycompounds comprising (i) unconjugated antibody monomer speciescomprising at least one disulfide bond formed by the reaction of twocysteine sulfhydryl moieties, (ii) unconjugated antibody dimer speciescomprising at least one disulfide bond formed by the reaction of twocysteine sulfhydryl moieties, and (iii) a combination of unconjugatedantibody monomer species and unconjugated antibody dimer species. Insome embodiments, the total concentration of unconjugated antibodycompounds compared to the area percentage of formed disulfide conjugatecompounds as measured by MS/LC is less than 10 area %, less than 5 area%, less than 4 area %, less than 3 area %, less than 2 area %, less than1 area %, less than 0.5 area %, less than 0.3 area %, less than 0.1 area%, or is not detectable.

IX. PBD Prodrug Methods of Treatment

It is contemplated that the PBD prodrug conjugate compounds of thepresent disclosure may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignantsolid tumors and hematological disorders such as leukemia and lymphoidmalignancies. Others include neuronal, glial, astrocytal, hypothalamic,glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory,angiogenic and immunologic, including autoimmune, disorders.

In one embodiment, a PBD prodrug conjugate compounds provided herein isused in a method of inhibiting proliferation of a cancer cell, themethod comprising exposing the cell to the antibody-prodrug conjugateunder conditions permissive for binding of the antibody orantibody-prodrug conjugates to a tumor-associated antigen on the surfaceof the cell, thereby inhibiting the proliferation of the cell. Incertain embodiments, the method is an in vitro or an in vivo method. Infurther embodiments, the cell is a lymphocyte, lymphoblast, monocyte, ormyelomonocyte cell.

Inhibition of cell proliferation in vitro may be assayed using theCellTiter-Glo™ Luminescent Cell Viability Assay, which is commerciallyavailable from Promega (Madison, Wis.). That assay determines the numberof viable cells in culture based on quantitation of ATP present, whichis an indication of metabolically active cells. See Crouch et al. (1993)J. Immunol. Meth. 160:81-88, U.S. Pat. No. 6,602,677. The assay may beconducted in 96- or 384-well format, making it amenable to automatedhigh-throughput screening (HTS). See Cree et al. (1995) AntiCancer Drugs6:398-404. The assay procedure involves adding a single reagent(CellTiter-Glo® Reagent) directly to cultured cells. This results incell lysis and generation of a luminescent signal produced by aluciferase reaction. The luminescent signal is proportional to theamount of ATP present, which is directly proportional to the number ofviable cells present in culture. Data can be recorded by luminometer orCCD camera imaging device. The luminescence output is expressed asrelative light units (RLU).

In another embodiment, a PBD prodrug conjugate compound for use as amedicament is provided. In further embodiments, a PBD prodrug conjugatecompound for use in a method of treatment is provided. In certainembodiments, a PBD prodrug conjugate compound for use in treating canceris provided. In certain embodiments, the disclosure provides a PBDprodrug conjugate compound for use in a method of treating an individualcomprising administering to the individual an effective amount of thePBD prodrug conjugate compound.

PBD prodrug conjugate compounds of the disclosure can be used eitheralone or in combination with other agents in a therapy. For instance, anantibody or immunoconjugate of the disclosure may be co-administeredwith at least one additional therapeutic agent. In some embodiments, theadditional therapeutic agent is an anthracycline. In some embodiments,the anthracycline is daunorubicin or idarubicin. In some embodiments,the additional therapeutic agent is cytarabine. In some embodiments, theadditional therapeutic agent is cladribine. In some embodiments, theadditional therapeutic agent is fludarabine or topotecan. In someembodiments, the additional therapeutic agent is 5-azacytidine ordecitabine.

Such combination therapies noted herein encompass combinedadministration (where two or more therapeutic agents are included in thesame or separate formulations), and separate administration, in whichcase, administration of the compounds of the disclosure can occur priorto, simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Compounds of the disclosure can alsobe used in combination with radiation therapy.

Compounds of the disclosure (and any additional therapeutic agent) canbe administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

Compounds of the disclosure would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners.Compounds of the disclosure need not be, but are optionally formulatedwith one or more agents currently used to prevent or treat the disorderin question. The effective amount of such other agents depends on theamount of the compound of the disclosure present in the formulation, thetype of disorder or treatment, and other factors discussed herein. Theseare generally used in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that is empirically/clinicallydetermined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of acompound of the disclosure (when used alone or in combination with oneor more other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of compound, the severity and course ofthe disease, whether the compound is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the compound, and the discretion of the attendingphysician. The compound is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of acompound of the disclosure can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned herein. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the compound would be in the range fromabout 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) maybe administered to the patient. Such doses may be administeredintermittently, e.g. every week or every three weeks (e.g. such that thepatient receives from about two to about twenty, or e.g. about six dosesof the antibody). An initial higher loading dose, followed by one ormore lower doses may be administered. However, other dosage regimens maybe useful. The progress of this therapy is easily monitored byconventional techniques and assays.

Intracellular release of the drug from the PBD prodrug conjugatecompound in a target cell is believed to result from a combination oflinker immolation and (i) GSH-activation of a disulfide trigger, (ii)DTD-activation of an quinone trigger or (iii) ROS-activation of an arylboronic acid or an aryl boronic ester trigger.

In connection with linkers comprising a disulfide moiety, GSH-mediatedrelease provides for advantages as compared to certain linkers known inthe prior art, such as acid-labile hydrazine linkers. More particularly,blood concentration of GSH is known to be very low, such as in themicromolar range, whereas intracellular GSH concentration is typicallyup to three orders of magnitude greater, such as in the millimolarrange. It is further believed that GSH concentration in cancer cells iseven greater due to increased activity of reductive enzymes. It is yetfurther believed that steric hindrance at the linker carbon atom bearinga sulfur atom provides for improved blood stream stability and improvedintracellular release. Therefore, it is believed that the disulfideconjugate compounds of the present disclosure provide for improvedstability in the bloodstream and for improved intracellular releaserates.

In connection with PBD prodrugs, GSH-, DTD- or ROS-activation of PBD N10protecting groups masks toxicity in the blood stream and in plasma andprovides for selective toxicity advantages as compared to PBD drugs notcomprising a protecting prodrug moiety. More particularly, bloodconcentration of GSH, DTD and ROS are known to be low as compared tocancer cells. Therefore, it is believed that the PBD prodrug conjugatecompounds of the present disclosure provide for reduced toxicity in thebloodstream and targeted intracellular activation to PBD.

X. Articles of Manufacture

In another embodiment of the disclosure, an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described herein is provided. The article ofmanufacture comprises a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe disorder and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is a PBD prodrug conjugate compound of thedisclosure. The label or package insert indicates that the compositionis used for treating the condition of choice. Moreover, the article ofmanufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises a PBD prodrugconjugate compound of the disclosure; and (b) a second container with acomposition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionor dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

EXAMPLES

The following are examples of methods and compounds of the disclosure.It is understood that various other embodiments may be practiced, giventhe general description provided herein.

The structures of PBD monomer and dimer compounds disclosed in theexamples are depicted below along with corresponding reference tagswherein the asterisk refers to a chiral center of racemic or undefinedstereochemistry.

Reference Tag Compound Structure PBD monomer control 1. Examples 6-9, 13and 15.

PBD monomer control 2. Examples 6, 9, 13 and 17.

PBD monomer disulfide prodrug 1. Examples 6, 9, 17 and 19K.

PBD monomer disulfide prodrug 2. Examples 6, 9, 17 and 19E.

PBD monomer disulfide prodrug 3. Examples 6, 9, 17 and 19Q.

PBD monomer disulfide prodrug 4. Examples 6, 9, 17 and 19Q.

PBD monomer disulfide prodrug 5. Examples 6, 9, 17 and 19R.

PBD monomer disulfide prodrug 6. Examples 6, 9 and 17.

PBD monomer disulfide prodrug 7. Examples 9, 17 and 19J.

PBD monomer disulfide prodrug 8. Examples 9 and 17.

PBD monomer disulfide prodrug 9. Examples 9 and 17.

PBD monomer disulfide prodrug 10. Examples 9, 17 and 19A.

PBD monomer disulfide prodrug 11. Examples 9, 17 and 19S.

PBD monomer disulfide prodrug 30. PBD monomer disulfide prodrug 11 andPBD monomer disulfide prodrug 30 are diastereomers having a differentconfiguration at one or more of the chiral centers designated with theasterisk. Examples 9, 17 and 19S.

PBD monomer disulfide prodrug 12. Examples 0, 17 and 19P.

PBD monomer disulfide prodrug 14. Example 17.

PBD monomer disulfide prodrug 15. Examples 9, 17 and 19C.

PBD monomer disulfide prodrug 16. Examples 9, 17 and 19F.

PBD monomer disulfide prodrug 13. Examples 9, 17 and 19D.

PBD monomer disulfide prodrug 17. Example 17.

PBD monomer disulfide prodrug 18. Example 17.

PBD monomer disulfide prodrug 19. Examples 6 and 17.

PBD monomer disulfide prodrug 20. Example 17.

PBD monomer disulfide prodrug 21. Example 17.

PBD monomer disulfide prodrug 22. Examples 9 and 17.

PBD monomer disulfide prodrug 23. Examples 9, 17 and 19O.

PBD monomer disulfide prodrug 24. Examples 9, 17 and 19N.

PBD monomer disulfide prodrug 25. Examples 9, 17 and 19M.

PBD monomer disulfide prodrug 26. Examples 9, 17 and 19L.

PBD monomer disulfide prodrug 27. Examples 9, 17 and 19I.

PBD monomer disulfide prodrug 28. Examples 9, 17 and 19H.

PBD monomer disulfide prodrug 29. Examples 9, 17 and 19G.

PBD Dimer control 1. Examples 7, 9, 15, 17 and 19E.

PBD Dimer control 2. Examples 9, 17 and 19F.

PBD Dimer Disulfide Prodrug 1. Examples 7, 9, 17, 20B and 21D.

PBD Dimer Disulfide Prodrug 4. Examples 7, 9, 20A and 21C.

PBD Dimer Disulfide Prodrug 2. Examples 9, 20C and 21B.

PBD Dimer Disulfide Prodrug 3. Examples 9, 20D and 21A.

Non-prodrug PBD dimer ADC controls 1 to 5, wherein an antibodysulfhydryl moiety is conjugated to the maleimide moiety Non-prodrug PBDdimer ADC control 1: 7C2 HC A140C (Her2). Examples 10 and 14.Non-prodrug PBD dimer ADC control 2: HC A118C (CD22). Examples 10 and13. Non-prodrug PBD dimer ADC control 3: 4D5 HC A118C (Her2). Examples10 and 14. Non-prodrug PBD dimer ADC control 4: 4D5 LC V205C (Her2).Example 10. Non-prodrug PBD dimer ADC control 5: 4D5 HC A118C (Her2).Example 16.

PBD dimer ADC disulfide prodrug 1A (Her2) and PBD dimer. Examples 10 and21D. ADC disulfide prodrug 1B (Examples 13 and 24D) each comprise a 7C2LC K149C (Her2) antibody conjugated to the maleimide moiety

PBD dimer ADC disulfide prodrug 2A wherein a 7C2 HC A140C (Her2)antibody is conjugated to the maleimide moiety (Examples 8, 10 and 21B)and PBD dimer ADC disulfide prodrug 2B wherein a 7C2 LC K149C (Her2)antibody is conjugated to the maleimide moiety (Examples 8, 10, 21 and21B)

PBD dimer ADC disulfide prodrug 3 wherein a 7C2 LC K149C antibody isconjugated to the maleimide moiety. Examples 8, 10 and 21A.

PBD dimer ADC disulfide prodrug 4 wherein a 7C2 LC K149C antibody isconjugated to the maleimide moiety. Examples 8, 10 and 21C.

PBD dimer ADC disulfide prodrug 5. Examples 8 and 21E.

PBD monomer boronic acid prodrug 1. Example 13.

PBD Dimer ADC boronic acid control 1A wherein a 10 F4v3; LC K149Cantibody is conjugated to the maleimide moiety (Examples 13 and 22B) PBDDimer ADC boronic acid control 1B wherein a Ly6 antibody; 9B12v.12; LCK149C is conjugated to the maleimide moiety (Example 22B)

PBD Dimer ADC boronic acid control 2 (positive control) wherein a LCK149C (CD22) antibody is conjugated to the maleimide moiety. Example 13.

PBD Dimer ADC boronic acid prodrug 1A wherein a LC K149C (CD22) antibodyis conjugated to the maleimide moiety (Examples 13, 14 and 22A) PBDDimer ADC boronic acid prodrug 1B wherein a LC K149C (Ly6E) antibody isconjugated to the maleimide moiety (Example 13 and 22A)

PBD Dimer Diaphorase prodrug 1. Examples 6, 15, 23B and 25.

PBD Monomer Diaphorase prodrug 2. Examples 6, 15, 18 and 23A.

PBD monomer diaphorase prodrug 3. Examples 6, 18 and 23C.

PBD dimer diaphorase prodrug 2. Examples 6 and 23D.

PBD dimer diaphorase prodrug 3. Examples 6 and 23E.

PBD dimer ADC diaphorase prodrug 1A wherein a 7C2 LC K149C antibody isconjugated to the maleimide moiety (Examples 10 and 25.) and PBD dimerADC diaphorase prodrug 1B wherein a Ly6E LC K149C antibody is conjugatedto the maleimide moiety (Example 8)

Example 1: General Method for ADC Preparation

Cysteine engineered antibodies, such as those listed in Tables A to D,were made reactive for conjugation with linker-drug intermediates insome aspects of the present disclosure by treatment with a reducingagent such as DTT (Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; see Getz et al (1999)Anal. Biochem. Vol 273:73-80 (incorporated herein by reference); SoltecVentures, Beverly, Mass.) followed by re-formation of the inter-chaindisulfide bonds (re-oxidation) with a mild oxidant such asdehydroascorbic acid. Full length, cysteine engineered monoclonalantibodies (THIOMAB™) expressed in CHO cells (see Gomez et al. (2010)Biotechnology and Bioeng. 105(4):748-760 (incorporated herein byreference); and Gomez et al (2010) Biotechnol. Prog. 26:1438-1445(incorporated herein by reference)) were reduced, for example, withabout a 50 fold excess of DTT overnight in 50 mM Tris, pH 8.0 with 2 mMEDTA at room temperature, which removes cysteine and GSH adducts as wellas reduces interchain disulfide bonds in the antibody. Removal of theadducts was monitored by reverse-phase liquid chromatography/massspectrometric Analysis (“LC/MS”) using a PLRP-S column. The reducedTHIOMAB™ was diluted and acidified by addition to at least four volumesof 10 mM sodium succinate, pH 5 buffer. Alternatively, the antibody wasdiluted and acidified by adding to at least four volumes of 10 mMsuccinate, pH 5 and titration with 10% acetic acid until pH wasapproximately five. The pH-lowered and diluted THIOMAB™ was subsequentlyloaded onto a HiTrap S cation exchange column, washed with severalcolumn volumes of 10 mM sodium acetate, pH 5 and eluted with 50 mM Tris,pH 8.0, 150 mM sodium chloride.

Disulfide bonds were reestablished between cysteine residues present inthe parent Mab by carrying out reoxidation. The eluted reduced THIOMAB™described above was treated with 15× dehydroascorbic acid (DHAA) forabout 3 hours or, alternatively, with 200 nM to 2 mM aqueous coppersulfate (CuSO4) at room temperature overnight. Other oxidants, i.e.oxidizing agents, and oxidizing conditions, which are known in the artmay be used. Ambient air oxidation may also be effective. This mild,partial reoxidation step formed intrachain disulfides efficiently withhigh fidelity. Reoxidation was monitored by reverse-phase LC/MS using aPLRP-S column. The reoxidized THIOMAB™ was diluted with succinate bufferas described above to reach pH approximately 5 and purification on an Scolumn was carried out as described above with the exception thatelution was performed with a gradient of 10 mM succinate, pH 5, 300 mMsodium chloride (buffer B) in 10 mM succinate, pH 5 (buffer A). EDTA wasadded to the eluted THIOMAB™ to a final concentration of 2 mM andconcentrated, if necessary, to reach a final concentration of more than5 mg/mL.

The resulting THIOMAB™, suitable for conjugation, was stored at −20° C.in aliquots. LC/MS Analysis was performed on a 6200 series TOF or QTOFAgilent LC/MS. Samples were chromatographed on a PRLP-S®, 1000 A,microbore column (50 mm×2.1 mm, Polymer Laboratories, Shropshire, UK)heated to 80° C. A linear gradient from 30-40% B (solvent A: 0.05% TFAin water, solvent B: 0.04% TFA in acetonitrile) was used and the eluentwas directly ionized using the electrospray source. Data were collectedand deconvoluted by the MassHunter software. Prior to LC/MS analysis,antibodies or drug conjugates (50 micrograms) were treated withPeptideN-Glucosidase F (“PNGase F”) (2 units/ml; PROzyme, San Leandro,Calif.) for 2 hours at 37° C. to remove N-linked carbohydrates.Alternatively, antibodies or drug conjugates were partially digestedwith LysC (0.25 ug per 50 ug antibody or conjugate) for 15 minutes at 37C to give a Fab and Fc fragment for analysis by LC/MS. Peaks in thedeconvoluted LC/MS spectra were assigned and quantitated.Drug-to-antibody ratios (DAR) were calculated by calculating the ratioof intensities of the peak or peaks corresponding to drug-conjugatedantibody relative to all peaks observed.

The THIOMAB™ for conjugation, in 10 mM succinate, pH 5, 150 mM NaCl, 2mM EDTA, was adjusted to pH 7.5-8.5 with 1M Tris. An excess, from about3 to 20 molar equivalents of a linker-drug intermediate of the presentdisclosure with a thiol-reactive pyridyl disulfide group was dissolvedin DMF or DMA and added to the reduced, reoxidized, and pH-adjustedantibody. The reaction was incubated at room temperature or 37° C. andmonitored until completion (1 to about 24 hours), as determined by LC/MSanalysis of the reaction mixture. When the reaction is complete, theconjugate may be purified by one or any combination of several methodsto remove remaining unreacted linker-drug intermediate and aggregatedprotein (if present at significant levels). For example, the conjugatemay be diluted with 10 mM histidine-acetate, pH 5.5 until final pH isapproximately 5.5 and purified by S cation exchange chromatography usingeither HiTrap S columns connected to an Akta purification system (GEHealthcare) or S maxi spin columns (Pierce). Alternatively, theconjugate may be purified by gel filtration chromatography using an S200column connected to an Akta purification system or Zeba spin columns.Alternatively, dialysis may be used. The THIOMAB™ drug conjugates wereformulated into 20 mM His/acetate, pH 5, with 240 mM sucrose usingeither gel filtration or dialysis. The purified conjugate isconcentrated by centrifugal ultrafiltration and filtered through a0.2-μm filter under sterile conditions and frozen for storage. Theantibody-drug conjugates were characterized by BCA assay to determineprotein concentration, analytical SEC (size-exclusion chromatography)for aggregation analysis and LC/MS after treatment with Lysine Cendopeptidase (LysC) to calculate DAR.

Size exclusion chromatography was performed on conjugates using a ShodexKW802.5 column in 0.2M potassium phosphate pH 6.2 with 0.25 mM potassiumchloride and 15% IPA at a flow rate of 0.75 ml/min. The aggregationstate of the conjugate was determined by integration of eluted peak areaabsorbance at 280 nm.

LC/MS analysis may be performed on conjugates using an Agilent QTOF 6520ESI instrument. As an example, the antibody-drug conjugate was treatedwith 1:500 w/w Endoproteinase Lys C (Promega) in Tris, pH 7.5, for 30min at 37° C. The resulting cleavage fragments were loaded onto a 1000 Å(Angstrom), 8 μm (micron) PLRP-S (highly cross-linked polystyrene)column heated to 80° C. and eluted with a gradient of 30% B to 40% B in5 minutes. Mobile phase A was H₂O with 0.05% TFA and mobile phase B wasacetonitrile with 0.04% TFA. The flow rate was 0.5 ml/min. Proteinelution was monitored by UV absorbance detection at 280 nm prior toelectrospray ionization and MS analysis. Chromatographic resolution ofthe unconjugated Fc fragment, residual unconjugated Fab and drugged Fabwas usually achieved. The obtained m/z spectra were deconvoluted usingMass Hunter™ software (Agilent Technologies) to calculate the mass ofthe antibody fragments.

Example 2: Herceptin A118C Antibody-Probe Conjugates

Various probe compounds comprising a linker and a thiol leaving groupwere conjugated with Herceptin A118C. In each conjugation, 5 mg/mLantibody in a solvent system was contacted with a probe-linker-leavinggroup compound at an equivalent ratio of probe compound to antibody of10:1 wherein the probe-linker is conjugated to the antibody via adisulfide bond. The solvent system comprised 75 mM Tris, pH 8.5 and 10v/v % DMF. The conjugation reaction was run at room temperature for 24hours. The reaction product mixture was analyzed by LC/MS to determine adrug to antibody ratio (DAR), an area percent of leaving group byproductas compared to antibody-probe conjugate, an area percent of unconjugateddimer as compared to antibody-probe conjugate, and an area percent ofunconjugated monomer as compared to antibody-probe conjugate.

The probe was of the formula below where the wavy line indicates thepoint of attachment to the linker:

Probe-linker-leaving group compounds evaluated included:

The results are reported in Table 1 below where “DAR” refers todrug-antibody ratio, “% LG” refers to percent leaving group byproduct,“% Unconj. Dimer” refers to percentage of unconjugated antibody dimerand “% Unconj. Monomer” refers to percentage of unconjugated antibodydimer.

TABLE 1 % Unconj. % Unconj. Linker-Leaving Group DAR % LG Dimer MonomerDimethyl/PDS 0.6 64 3 6 Dimethyl/5-nitroPDS 0.7 35 22 1Dimethyl/3-nitroPDS 0.2 69 18 2 Dimethyl/Ellman 0.2 89 2 1Cyclopropyl/5-nitroPDS 2 1 0 0 Ethyl/5-nitroPDS 2 1 0 0 Methyl/PDS 1.139 0 3 Methyl/5-nitroPDS 1.4 27 1 1 Methyl/3-nitroPDS 2 2 0 0Methyl/Ellman 1.7 17 1 0

Example 3: Herceptin K149C Antibody-Probe Conjugates

The probe compounds of Example 2 were evaluated for conjugation toHerceptin K149C via a disulfide bond under the reaction conditions ofExample 2. The reaction product mixture was analyzed by LC/MS todetermine a drug to antibody ratio (DAR) and an area percent of leavinggroup byproduct as compared to antibody-probe conjugate. The results arereported in Table 2 below.

TABLE 2 Linker-Leaving Group DAR % LG Dimethyl/PDS 0.3 84Dimethyl/5-nitroPDS 0.6 68 Dimethyl/3-nitroPDS 0.2 92 Dimethyl/Ellman0.2 91 Cyclopropyl/5-nitroPDS 2 0.4 Ethyl/5-nitroPDS 2 0.3 Methyl/PDS0.7 62 Methyl/5-nitroPDS 1.6 20 Methyl/3-nitroPDS 1.8 9 Methyl/Ellman1.8 9

Example 4: Herceptin A118C ADC

Various drug compounds comprising a linker and a 5-nitroPDS thiolleaving group were conjugated via a disulfide bond with Herceptin2 4D5HC A118C antibody. In each conjugation, 5 mg/mL antibody in a solventsystem was contacted with a drug-linker-leaving group compound at anequivalent ratio of drug-linker-leaving group compound to antibody of3:1. The solvent system comprised 75 mM Tris, pH 8.5. The conjugationreaction was run at room temperature for 3 hours. The reaction productmixture was analyzed by LC/MS to determine a drug to antibody ratio(DAR) and an area percent of leaving group byproduct as compared toantibody-probe conjugate.

The drug was of the formula below where the wavy line indicates thepoint of attachment to the linker:

Drug-linker-leaving group compounds evaluated included where “D” denotesa drug:

The results are reported in Table 3 below.

TABLE 3 Linker-Leaving Group DAR % LG Unsubstituted 1.9 2.8 Cyclopropyl1.9 4.8 Cyclobutyl 1.9 5 Cyclopentyl 0.8 48 Dimethyl 0.4 71 Cyclohexyl0.3 54 THP 0 96 Cyclohexyl-PAB 0 10 with visible precipitate

Example 5: xCD22 K149C ADC

A linker-drug compound comprising a methylsulfonate leaving group (MTS)(Linker 1) was conjugated via a disulfide bond with xCD22 K149C antibodyin a first evaluation. In the second evaluation, a linker-drug compoundcomprising a MTS leaving group (Linker 2) was conjugated with antibodyin a second evaluation. In each conjugation, 5 mg/mL antibody in asolvent system was contacted with a drug-linker-leaving group compoundat an equivalent ratio of probe compound to antibody of 3:1. The solventsystem comprised 75 mM Tris, pH 8.5. The conjugation reactions were runat room temperature for 3 hours. Linkers 1 and 2 are illustrated below:

The reaction product mixture was analyzed by LC/MS to determine a drugto antibody ratio (DAR) and an area percent of leaving group byproductas compared to antibody-probe conjugate. For Linker 1, the drug toantibody ratio was 2.0, the area percent of the antibody-drug conjugatewas about 91%, the area percent of unconjugated antibody was less than0.1%, and the area percent of MTS byproducts was about 0.5%. For Linker2, the drug to antibody ratio was 1.5, the area percent of theantibody-drug conjugate was about 91%, the area percent of unconjugatedantibody was less than 0.1%, and the area percent of MTS byproducts wasabout 0.5%.

Example 6: Toxicity of PBD Monomer Disulfide Prodrugs Against KPL-4 andWSU-DLCL2 Cell Lines

The toxicity of PBD monomer disulfide prodrugs and diaphorase prodrugswere evaluated against KPL-4, WSU-DLCL2, HCT-116, HCC1395 and Jurkatcell cultures. KPL-4 (a breast cancer cell line) expresses Her2 andexhibits high GSH levels of from about 12 to about 24 mM. KPL-4 cellsfurther have a high expression of DTD on the order of about 839 nRPKM(where nRPKM refers to normalized reads per Kb of transcript length permillion mapped read). WSU-DLCL2 (a non-Hodgkin's lymphoma cell line)exhibits lo GSH levels of about 1.4 mM and low DTD expression of about1.4 nRPKM.

Potency of the disulfide and DT-diaphorase prodrugs was measured by acell proliferation assay employing the following protocol (CELLTITERGLO™ Luminescent Cell Viability Assay, Promega Corp.). First, an aliquotof 40 ul of cell culture containing about 8000 cells of WSU-DLCL2 cells(low NQO1 gene and GSH level), 4000 cells of KPL-4 (high NQO1 gene andGSH level), HCT-116 cells, HCC1395 cells or Jurkat cells in RPMI-1640culture medium (supplemented with 10% fetal bovine serum, 2 mMglutamine, 50 uM cystine, and 0.015 g/L L-methionine) was deposited ineach well of a 384-well flat clear bottom white polystyrene tissueculture-treated microplates (Corning, N.Y.). Second, control wells wereprepared containing medium with and without cells. Third, compounds withand without disulfide or DT-diaphorase prodrugs (n=3) were added to theexperimental wells using ECHO acoustic liquid handling technology(Labcyte Inc, Sunnyvale, Calif.) to create a 10-point dose-responsecurve in triplicate with 1:3× serial dilution. Fourth, cells werecultured in a humidified incubator set at 37° C. and maintaining anatmosphere of 5% CO₂. Fifth, the plates were equilibrated to roomtemperature for approximately 30 minutes. Sixth, a volume of CELLTITERGLO™ Reagent equal to the volume of cell culture medium present in eachwell was added. Seventh, the contents were mixed for 10 minutes on anorbital shaker in the dark to induce cell lysis. Eighth, the plate wasincubated at room temperature for 30 minutes to stabilize theluminescence signal. Ninth, luminescence was recorded and reported ingraphs as % activity where RLU (relative luminescence units) wasnormalized to controls (no compound control minus no cell control).Tenth, data was plotted as individual points for each replicate (n=3)for each antibody as the mean of luminescence for each set ofreplicates, with standard deviation error bars.

The IC₅₀ potency results are presented in Table 4 below in nM whereinthe data in parenthesis represent IC₅₀ results for repeat evaluations:

TABLE 4 WSU- PBD agent KPL-4 DLCL2 HCT-116 HCC1395 Jurkat PBD monomer330.1 (240.63) 105.5 (108.7) — — — control 1 PBD monomer >10,000 >10,000— — — control 2 PBD monomer 323.8 274 — — — disulfide prodrug 1 PBDmonomer 497.3 1015.7 — — — disulfide prodrug 2 PBD monomer 359.7 530.6 —— — disulfide prodrug 3 PBD monomer >10,000 >10,000 — — — disulfideprodrug 4 PBD monomer 1057.6 1349.7 — — — disulfide prodrug 5 PBDmonomer 805.7 2806.5 — — — disulfide prodrug 6 PBD monomer 343.8 (354.9)574.7 (287.5) 223   — — diaphorase prodrug 2 PBD monomer 350.8 443.1 — —— diaphorase prodrug 3 PBD dimer 69.2 (12.9) 17.1 (1.77) 21.3 14.2 2.05diaphorase prodrug 1 (45.6) (3.67) PBD dimer 10.1 0.8 —  9.84 1.25diaphorase prodrug 2 PBD dimer 1.87 0.24  1.23 — — diaphorase prodrug 3

Results against KPL-4 for PBD monomer control 1 and PBD monomerdisulfide prodrugs 1 to 4 are depicted in FIG. 1 and results againstWSU-DLCL2 for PBD monomer control 1 and PBD monomer disulfide prodrugs 1to 4 are depicted in FIG. 2 where each figure is a plot of Activity [%]versus the log of prodrug concentration in moles per liter. Activity [%]is reported in negative values that refer to the reduction in cellviability. For instance, an Activity [%] of −50 refers to a 50%reduction in cell viability. Excluding Prodrug 4, for KPL-4, forprodrugs 1 to 3, the ratio of the parent compound to the prodrug havingthe greatest IC₅₀ (prodrug 2) was about 1.5. Excluding Prodrug 4, forWSU-DLDL2, for prodrugs 1 to 3, the ratio of the parent compound to theprodrug having the greatest IC₅₀ (prodrug 2) was about 9.6. Differentialdisulfide prodrug activation was observed in both the high GSH cell line(KPL-4) and the low GSH cell line (WSU-DLCL2). Less differentiation wasobserved for the KPL-4 cell line as compared to the WSU-DLCL2 cell line.

In addition to the potency evaluation, the release of PBD dimerdiaphorase prodrug 1 was evaluated. The release was measured to be 12%after 90 minutes incubation according to the following method. First, amaster mix reaction was prepared containing 0.5 uM of the PBD prodrug,50 uM PBD, 200 mM NADPH was quenched and diluted with 40 uL of 0.1%formic acid in 96-well polypropylene plate. Second, the reaction mix wasincubated at room temperature for 5, 30, 90 min. Third, standards inDMSO were prepared in 0.1% formic acid using ECHO acoustic liquidhandling technology (Labcyte Inc, Sunnyvale, Calif.) to create a10-point dose-response curve in triplicate with 1:2.5× serial dilutionin 96-well polypropylene plate. Fourth, 10 uL of sample and standard wasinjected to an AB SCIEX QTRAP® 6500 mass spectrometer coupled withWaters liquid chromatography. The LC gradient used was PhenomenexKinetex C18, 1.7 μm, 100 Å, 100×2.1 mm column, with mobile phase A (0.1%formic acid in water) and B (0.1% formic acid in acetonitrile) was 0-0.5min 5% B, 1-1.10 min 70-95% B, 2.49-2.5 min 95% B, 2.5-3.0 min 95-5% Bat a flow rate of 0.8 mL/min (column temperature of 30° C.). Theretention time was 1.06 min. The multiple reaction monitor (MRM)transition in MS was (585/257.0) and (585/504.2). The compound-dependentMS parameters for MRM (585/257.0) are 51, 20, 206, and 10 for CE, CXP,DP, and EP, respectively and for MRM (585/504.2) are 27, 12, 206, and 10for CE, CXP, DP, and EP, respectively. Finally, data was analyzed usingMultiQuant analysis software and standards were calculated usingGraphPad Prism 6 linear regression fit.

Example 7: Toxicity of PBD Monomer Disulfide Prodrugs Against SK-BR-3Cell Line

The toxicity of PBD monomer disulfide prodrugs 1 to 4, PBD dimerdisulfide prodrugs 1 and 4, PBD monomer control 1 and PBD dimer control1 were evaluated against SK-BR-3 cell cultures. The SK-BR-3 cell line isa breast adenocarcinoma that overexpresses HER2 and exhibits GSH levelsof from about 3 to about 10 mM. The HER2-positive breast cancer cellline, SK-BR-3, was obtained internally at Genentech after having beenverified by sequence analysis. The cells were grown at 37 Celsius inDMEM+10% FBS supplemented with 2 mM L-glutamine for the duration of thisassay. On the first day of the experiment, designated “Day −1”, SK-BR-3cells were harvested from tissue culture flasks, counted, and thenseeded into 96-well plates at a concentration of 7,500 cells per well.The next day, designated “Day 0”, cell culture media was removed fromthe cells and replaced with 100 uL of fresh media containing a serialdilution (1:3) of the compounds in Table 5 below. The plates were thenreturned to the 37 Celsius incubator and allowed to incubate for 3 days.On “Day 3” of the assay, the plates were removed from the incubator andallowed to equilibrate to room temperature before adding 100 uL ofroom-temperature CellTiter-Glo reagent (Promega). The plates were thenagitated at 300 rpm for 10 minutes. The luminescence signal generatedfrom this reaction was recorded on an Envision plate reader(Perkin-Elmer). The data were arranged in Excel (Microsoft) and graphedin Kaleidagraph (Synergy Software).

The IC₅₀ potency results are presented in Table 5 below:

TABLE 5 SK-BR-3 PBD agent IC₅₀ (nM) PBD Monomer Control 1 124 PBDMonomer Disulfide Prodrug 1 331 PBD Monomer Disulfide Prodrug 3 616 PBDMonomer Disulfide Prodrug 2 1757 PBD Monomer Disulifde Prodrug 4 >10,000PBD Dimer Control 1 0.144 PBD Dimer Disulfide Prodrug 1 2.39 PBD DimerDisulfide Prodrug 4 3.09

The results for SK-BR-3 are depicted in FIGS. 3 and 10 where the figuresare plots of Cell viability (% of control) versus PBD compoundconcentration on an nM basis. Based on the results, it is believed thatbulkier disulfide prodrug moieties have reduced potency. Further, basedon experimental results to date, it is believed that potency correlateswith disulfide stability.

Example 8: Whole Blood Stability of PBD Monomer and Dimer Prodrugs

The whole blood stability of various PBD monomer and PBD dimer prodrugsof the present invention was evaluated.

Whole blood stability samples were prepared as follows. Stabilitysamples were generated in Mouse (CB 17 SCID), Rat (Sprague-Dawley),Cynomologus Monkey and Human Whole blood Plasma as well as Buffer (0 and24 hour). Blood was collected by Bioreclamation then shipped coldovernight and samples were created immediately on arrival of wholeblood. To create stability samples, initial dilutions of the sourcematerial were made in Buffer (1×PBS, 0.5% BSA, 15 PPM Proclin) so thatall molecules were 1 mg/mL in concentration. Then a 1:10× dilution (36uL of 1 mg/mL initial dilution+324 uL blood or buffer) was performed togenerate the stability samples with a final concentration of 100 ug/mL.Once mixed, 150 μL of the Whole Blood/Buffer stability samples wasaliquoted into two separate sets of tubes for the two different timepoints. The 0 hour time points were then placed in a −80° C. freezer,while the 24 hour time points were placed on a shaker in a 37° C.incubator. When the 24 hour samples reached the given time point theywere also placed in the −80° C. freezer.

The whole blood stability samples were evaluated by affinity-captureLC-MS assay. First, Streptavidin-coated magnetic beads (LifeTechnologies Corporation, Grand Island, N.Y.) were washed 2× with HBS-EPbuffer (GE Healthcare, Sunnyvale, Calif.), then mixed with biotinylatedCD22 anti-idiotypic antibody using the KingFisher Flex (Thermo FisherScientific, Waltham, Mass.) and incubated for 2 hrs at room temperaturewith gentle agitation. After the 2 hrs, the SA-bead/Biotin-xId Abcomplex was washed 2× with HBS-EP buffer, mixed with the diluted wholeblood stability samples and then incubated for 2 hrs at room temperaturewith gentle agitation. After the 2 hrs, the SA-bead/Biotin-xId Ab/samplecomplex was washed 2× with HBS-EP buffer, mixed with the deglycosylationenzyme PNGase F (New England BioLabs, Ipswich, Mass.) and then incubatedfor overnight at 37° C. with gentle agitation. After the overnightincubation, the deglycosylated SA-bead/Biotin-xId Ab/sample complex waswashed 2× with HBS-EP buffer, followed by 2× washes of water (OptimaH₂O, Fisher Scientific, Pittsburgh, Pa.) and finally 1× wash with 10%acetonitrile. The beads were then placed in 30% acetonitrile/0.1% formicacid for elution where they incubated for 30 mins at room temperaturewith gentle agitation before the beads were collected. The elutedsamples were injected and loaded onto a Thermo Scientific PepSwift RPmonolithic column (500 μm×5 cm) maintained at 65° C. The sample wasseparated on the column using a Waters Acquity UPLC system at a flowrate of 20 μL/min with the following gradient: 20% B (95%acetonitrile+0.1% formic acid) at 0-2 min; 35% B at 2.5 min; 65% B at 5min; 95% B at 5.5 min; 5% B at 6 min. The column was directly coupledfor online detection with a Waters Synapt G2-S Q-ToF mass spectrometryoperated in positive ESI with an acquisition mass range from 500 to 5000Th (m/z).

The in vitro whole blood stability of PBD monomer disulfide prodrugs 2to 6, 8, 10 and 13, and PBD monomer control 1 were evaluated in humansand rats at 4- and 24-hour intervals. The results are presented in FIG.4 as percent of the parent compound remaining relative to time zero.

The in vitro whole blood stability of PBD dimer ADC boronic acid prodrug1, PBD dimer ADC diaphorase prodrug 1B, and PBD dimer ADC disulfideprodrugs 1, 2A, 2B and 3 to 5 were evaluated in mouse, rat, cyno andhuman and the results are presented in Table 6 below where “DAR” refersto drug to antibody ratio, “Delta % DAR2” refers to delta DAR2 at 24hours relative to the buffer at 0 hours, and “Prodrug loss” refers toprodrug loss at 24 hours relative to the buffer at 0 hours.

TABLE 6 % DAR2 % DAR2 Delta Prodrug Prodrug Matrix 0 hr 24 hr % DAR2loss PBD dimer ADC Buffer 100 100 0 0.23 disulfide Mouse 100 100 0 3.00prodrug 1 Rat 100 100 0 5.60 Cyno 100 100 0 4.23 Human 89 89 11 5.07 PBDdimer ADC Buffer 100 100 0 0 disulfide Mouse 94.89 95.18 4.82 61.97prodrug 2A Rat — 100 0 72.09 Cyno 93.94 100 0 91.01 Human 94.33 100 055.15 PBD dimer ADC Buffer 100 100 0 0 disulfide Mouse 100 100 0 61.42prodrug 2B Rat 100 100 0 78.41 Cyno 100 100 0 88.20 Human 86 90 10 45.89PBD dimer ADC Buffer 100 100 0 0 disulfide Mouse 100 100 0 83.22 prodrug3 Rat 100 100 0 87.21 Cyno 100 100 0 92.20 Human 84 80 20 79.74 PBDdimer ADC Buffer 100 100 0 0 disulfide Mouse 100 100 0 32.47 prodrug 4Rat 100 100 0 21.42 Cyno 100 100 0 36.25 Human 89 85 15 25.30 PBD dimerADC Buffer 100 100 0 0.15 disulfide Mouse 100 100 0 57.66 prodrug 5 Rat100 100 0 81.35 Cyno 100 100 0 97.36 Human 100 100 0 67.90 PBD dimer ADCBuffer 100 100 0 0 boronic acid Mouse 100 100 0 0 prodrug 1 Rat 100 1000 0 Cyno 100 100 0 0 Human 100 100 0 0 PBD dimer ADC Buffer 100 100 00.71 diaphorase Mouse 100 100 0 11.75 prodrug 1B Rat 100 100 0 11.32Cyno 100 100 0 4.67 Human 100 100 0 2.30

Example 9: Toxicity of PBD Monomer Disulfide Prodrugs and PBD DimerDisulfide Prodrugs Against Various Cell Lines

The toxicity of various PBD monomer disulfide prodrugs and PBD dimerdisulfide prodrugs was evaluated on UACC-257, Igrov-1 and A2058 celllines. Cells were seeded in 384-well plate and treated with drug 24hours later. After 4 days of drug incubation, the cell viability wasdetermined using Promega CellTiter-Glo luminescent reagent, whichmeasures ATP level (an indirect measure of cell number). The luminescentintensity was measured on PerkinElmer Envision reader. The relative cellviability was calculated by normalizing to non-drug treatment controland was graphed using KleidaGraph software package. IC₅₀ value wasdetermined as the concentration to obtain 50% of the maximum cellkilling.

The IC₅₀ results are presented in Table 7A below:

TABLE 7A PBD Agent UACC-257 Igrov-1 A2058 PBD monomer control 1 181.468.5 56.9 PBD monomer control 2 >20,000 >20,000 >20,000 PBD monomerdisulfide prodrug 1 275.7 122.8 82.9 PBD monomer disulfide prodrug 21858.8 1871.8 635.2 PBD monomer disulfide prodrug 3 356.0 253.9 75.2 PBDmonomer disulfide prodrug 4 19164.1 12640.1 8109.9 PBD monomer disulfideprodrug 5 892.1 765.1 395.3 PBD monomer disulfide prodrug 6 497.4 3262.3566.6 PBD monomer disulfide prodrug 7 >30,000 >30,000 10956 PBD monomerdisulfide prodrug 8 194.1 171.8 47.2 PBD monomer disulfide prodrug 92109.1 1046.9 291.8 PBD monomer disulfide prodrug 10 4732.3 4807.61827.9 PBD monomer disulfide prodrug 11 331.1 206.7 108.3 PBD monomerdisulfide prodrug 12 155.8 83.6 31.1 PBD monomer disulfide prodrug13 >10,000 >10,000 >10,000 PBD monomer disulfide prodrug 15 1500.2 928.2304.2 PBD monomer disulfide prodrug 16 19460.1 18509.1 5593.4 PBDmonomer disulfide prodrug 19 147.3 77.4 37.7 PBD monomer disulfideprodrug 22 80.6 39.8 23.7 PBD monomer disulfide prodrug 23 866.2 687.2206.3 PBD monomer disulfide prodrug 24 2694.2 1479.9 596.1 PBD monomerdisulfide prodrug 25 241.6 129.4 47.7 PBD monomer disulfide prodrug 26328.3 245.8 84.5 PBD monomer disulfide prodrug 27 939.1 957.9 261.2 PBDmonomer disulfide prodrug 28 2102.2 2304.1 920.1 PBD monomer disulfideprodrug 29 142.1 57.8 29.1 PBD monomer disulfide prodrug 30 307.6 234.8107.6 PBD dimer control 1 (in GSH cell panel) 0.95 0.061 — PBD dimerdisulfide prodrug 1 2.2 0.21 — PBD dimer disulfide prodrug 4 7.8 2.5 —

Note that an IC₅₀ ratio may be calculated for each prodrug as comparedto the respective control. For instance, The IC₅₀ ratio of PBD dimerdisulfide prodrug 1 is 2.3 and 3.4 for cell lines UACC-257 and IGROV-1,respectively, calculated as follows: (PBD dimer disulfide prodrug 1UACC-257 IC₅₀ of 2.2)/(PBD dimer control 1 UACC-257 IC₅₀ of 0.95)=Ratioof 2.3); and (PBD dimer disulfide prodrug 1 IGROV-1 IC₅₀ of 0.21)/(PBDdimer control 1 IGROV-1 IC₅₀ of 0.061)=Ratio of 3.4).

PBD dimer control 1 and PBD dimer control 2 were further evaluated instandard cell panels against a number of cell lines. The IC50 resultsare reported in Table 7B below.

TABLE 7B Cell line PBD dimer control 1 PBD dimer control 2 MES-SA 0.02810.0 MES-SA/Dx5 0.56 >100 BJAB 0.015 4.9 BJAB/Pgp — 53.7 KPL-4 0.05367.4 HCC1569 X2 0.097 — T-47D 0.032 — HCC1937 0.15 — NCI-H1781 0.011 —SW 900 0.078 — MDA-MB-231 — 73.2 HCT 116 — 15.0 A2058 — 5.3 DLD-1 — >100HL-60 — 2.4

The PBD monomer disulfide prodrugs 2, 3, 5, 6, PBD dimer disulfideprodrug 1 to 3, PBD monomer control 1 and PBD dimer control 1 resultsfor SK-BR-3 are depicted in FIG. 5 and the results for KPL-4 aredepicted in FIG. 6 where each figure is a plot of Cell viability (% ofcontrol) versus PBD compound concentration in Ig/mL. Differentialdisulfide prodrug activation was observed in both the high GSH cell line(KPL-4) and the low GSH cell line (WSU-DLCL2). Less differentiation wasobserved for the KPL-4 cell line as compared to the WSU-DLCL2 cell line.

The results for the PBD dimer control 1 against UACC-257 and IGROV-1cell lines is depicted in FIG. 7. The results for PBD dimer disulfideprodrug compound 1 against UACC-257 and IGROV-1 cell lines are depictedin FIG. 8. The results for PBD dimer disulfide prodrug compound 4against UACC-257 and IGROV-1 cell lines is depicted in FIG. 9.

Example 10; Toxicity of PBD Dimer Disulfide Prodrug-Antibody ConjugatesAgainst SK-BR-3 and KPL-4 Cell Lines

The toxicity of various PBD dimer ADC disulfide prodrugs, PBD dimer ADCdiaphorase prodrugs, and non-prodrug PBD dimer ADC controls wereevaluated against KPL-4 and SK-BR-3 cell cultures. Cells were plated inblack-walled 96-well plates (4000 per well for SK-BR-3; 1200 cells perwell for KPL-4) and allowed to adhere overnight at 37° C. in ahumidified atmosphere of 5% CO₂. Medium was then removed and replaced byfresh culture medium containing different concentrations of ADCs. After5 days, Cell Titer-Glo reagent was added to the wells for 10 min at roomtemperature and the luminescent signal was measured using PerkinElmerEnVision.

The IC₅₀ results are presented in Table 8 below:

TABLE 8 SK-BR-3 IC₅₀ KPL-4 IC₅₀ PBD agent (ng/mL) (ng/mL) PBD dimer ADCdisulfide prodrug 3 7.0 5.7 PBD dimer ADC disulfide prodrug 2B 8.9 31.7PBD dimer ADC disulfide prodrug 2A 8.4 (14) 5.0 (10) PBD dimer ADCdisulfide prodrug 4 19.3 247 PBD dimer ADC disulfide prodrug 1A 16.133,000 PBD dimer ADC disulfide prodrug 1B 11.1 (18.5) 8.8 (17.6)Non-prodrug PBD dimer ADC control 1 0.6 0.5 Non-prodrug PBD dimer ADCcontrol 2 349 333 Non-prodrug PBD dimer ADC control 3 4.1 1.3Non-prodrug PBD dimer ADC control 4 1.4 >100,000 PBD dimer ADCdiaphorase prodrug 1A 8.0 5.4 PBD dimer ADC diaphorase prodrug 1B >1,000<1,000

The PBD dimer ADC disulfide prodrug 2A, PBD dimer ADC disulfide prodrug1B, non-prodrug PBD dimer ADC control 1 and non-prodrug PBD dimer ADCcontrol 2 results for SK-BR-3 are depicted in FIG. 11 and the resultsfor KPL-4 are depicted in FIG. 12 where each figure is a plot of Cellviability (% of control) after 5 days versus PBD compound concentrationin μg/mL. Differential disulfide prodrug activation was observed withHER2 conjugates in HER2 cell lines.

Example 11: Toxicity of PBD Dimer Disulfide Prodrug-Antibody ConjugatesAgainst SK-BR-3 and KPL-4 Cell Lines

The toxicity of PBD dimer ADC disulfide prodrugs 1, 2B, 3 and 4 andnon-prodrug PBD dimer ADC controls 1 and 2 were evaluated againstSK-BR-3 and KPL-4 cell lines for cell viability after 5 days. Cells wereplated in black-walled 96-well plates (4000 per well for SK-BR-3; 1200cells per well for KPL-4) and allowed to adhere overnight at 37° C. in ahumidified atmosphere of 5% CO₂. Medium was then removed and replaced byfresh culture medium containing different concentrations of ADCs. After5 days, Cell Titer-Glo reagent was added to the wells for 10 min at roomtemperature and the luminescent signal was measured using PerkinElmerEnVision.

The results are reported in FIGS. 13 and 14.

Example 12: Whole Blood Stability of PBD Dimer-Antibody ConjugateDisulfide Prodrugs

The stability of PBD dimer ADC disulfide prodrugs 1 and 2B wereevaluated in a buffer (“Buffer”), in cynomolgus monkey whole blood(“CynoWB”), in human whole blood (“HumanWB), in mouse whole blood(“MouseWB”), and in rat whole blood (“RatWB”). The experimental protocolis described in Example 8. The results are reported in Table 9 belowwhere percent loss refers to the total loss of prodrug as compared theprodrug concentration at time zero. Loss of prodrug includes loss of theimine form (i.e., a hydroxyl moiety at C11 adjacent to the N10 prodrugsubstitution point).

TABLE 9 PBD dimer ADC disulfide PBD dimer ADC disulfide prodrug 1prodrug 2B Buffer  5% loss  5% loss CynoWB 10% loss 90% loss HumanWB 10%loss 40% loss MouseWB  8% loss 63% loss RatWB 10% loss 80% loss

Example 13: Toxicity of a ROS-Activated PBD Dimer-Antibody ConjugateAryl Boronic Acid Prodrugs

The toxicity of various PBD monomer and dimer boronic acid prodrugcompounds were evaluated for cell toxicity. In the in vitro tumor cellkilling assay, the cells were added to each well of 96-well microtiterplates at 8,000 cells per well and were incubated overnight at 37° C. ina humidified atmosphere of 5% CO₂. The cells were exposed to variousconcentration of the indicated prodrug, positive control and negativecontrol compounds. Where silvestrol was evaluated, a 1:3 serial dilutionwas used. After incubation for 3 days, Cell titer-Glo reagent (Promega,Madison Wis.) was added to the wells at 100 μL per well followed by a10-minute incubation at room temperature, and the luminescent signal wasmeasured using a Packard/Perkin-Elmer TopCount.

The toxicity of PBD dimer ADC boronic acid prodrug 1A and 1B wereevaluated against WSU-DLCL and BJAB tumor cell lines for cell viability.PBD dimer ADC boronic acid control 1A was used as a negative control andPBD dimer ADC boronic acid control 2 was used as a positive control. Theresults are reported in FIGS. 15A, 15B and 16 as normalized percent ofviable cells after 3 days as compared to the number of cells at timezero versus dosage in μg/mL. PBD dimer ADC boronic acid prodrug 1Aprovided an EC₅₀ of 1.701 nM and the non-prodrug PBD dimer ADC control 2provided an EC₅₀ of 0.0236 nM. PBD dimer ADC boronic acid prodrug 1Aprovided an EC₅₀ of 0.0509 nM, PBD dimer ADC boronic acid prodrug 1Bprovided an EC₅₀ of 15.86 nM, and PBD dimer ADC boronic acid control 2provided an EC₅₀ of 0.0274.

The toxicity of PBD monomer boronic acid prodrug 1 was evaluated againstMDA-MB-453 tumor cell line for cell viability. PBD monomer control 1 wasused as a positive control and PBD monomer control 2 was used as anegative control. The toxicity study was repeated wherein the PBDprodrug and controls were administered in combination with silvestrol.FIG. 15C depicts a plot of MDA-MB-453 cell kill versus drugconcentration in pM three days after exposure to: (i) a PBD monomercontrol; (ii) a PBD monomer control having a benzyl formate moiety atthe N10 PBD position; and (iii) a PBD monomer boronic acid prodrug. FIG.15D depicts a plot of MDA-MB-453 cell kill versus drug concentration inμM three days after exposure to: (i) silvestrol, (ii) a PBD monomercontrol; (iii) a PBD monomer control having a benzyl formate moiety atthe N10 PBD position; (iv) a PBD monomer boronic acid prodrug; (v) thePBD monomer control and silvestrol; (vi) the PBD monomer control havinga benzyl formate moiety at the N10 PBD position and silvestrol; and(vii) the PBD monomer boronic acid prodrug and silvestrol. PBD monomercontrol 1 provided an EC₅₀ of 0.1635 nM, PBD monomer boronic acidprodrug 1 provided an EC₅₀ of 1.846 nM, and PBD monomer prodrug 2provided an EC₅₀ of 267,356 nM.

The results indicate that the ROS aryl boronic acid prodrug provided forincreased cell kill relative to the negative control.

Example 14: Efficacy of Anti-CD22 and Anti-Her2 Antibody Drug Conjugates

The efficacy of the anti-CD22 antibody-drug conjugates (ADCs) wasinvestigated in a mouse xenograft model of BJAB-luc human Burkitt'slymphoma. The BJAB-luc cell line was obtained from Genentech cell linerepository. This cell line was authenticated by short tandem repeat(STR) profiling using the Promega PowerPlex 16 System and compared withexternal STR profiles of cell lines to determine cell line ancestry. TheBJAB-luc cell line expresses CD22 as determined by FACS and IHC. Toestablish the xenograft model, female C.B-17 SCID mice (Charles RiverLaboratories) were each inoculated subcutaneously in the flank area withthe BJAB-luc cells (20 million cells suspended in 0.2 mL Hank's BalancedSalt Solution; Invitrogen).

The efficacy of the anti-Her2 antibody-drug conjugates (ADCs) wasinvestigated in a mouse xenograft model of KPL4 human breast cancer. TheKPL4 cell line was obtained from Dr. J. Kurebayashi lab (Japan) and thiscell line expresses HER2 as determined by FACS and IHC. To establish thexenograft model, female C.B-17 SCID-beige mice (Charles RiverLaboratories) were each inoculated in the thoracic mammary fat pad areawith the KPL4 cells (3 million cells suspended in 0.2 mL of 1:1 mixtureof Hank's Balanced Salt Solution; Invitrogen and Matrigel; BDBiosciences). The KPL4 xenograft is a cachexia-inducing model whereanimals lost about 5% of their initial body weight in response to thetumor itself. Administration of anti-Her2 ADC attenuated this tumorassociated weight loss, and was well tolerated in the animals.

When tumors reached an average tumor volume of 100-300 mm³, the animalswere randomized into groups of 5-10 mice each and received a singleintravenous injection of the ADCs (referred to as Day 0). In a firstevaluation as depicted in FIGS. 23 and 24, mice were treated with thevehicle (histidine buffer #8, 100 μL IV once), non-prodrug PBD dimer ADCcontrol 2 (0.1 mg/kg and 0.2 mg/kg IV once), PBD dimer ADC boronic acidprodrug 1A (0.2, 0.5, 1, 2, and 5 mg/kg IV once), PBD dimer ADC boronicacid control 1A (1 mg/kg IV once), PBD dimer ADC boronic acid prodrug 1B(1 mg/kg IV once) and non-prodrug PBD dimer ADC control 3 (0.2 and 1mg/kg IV once). In a second evaluation depicted in FIGS. 25 and 26, micewere treated with the vehicle (histidine buffer #8, 100 μL IV once),non-prodrug PBD dimer ADC control 1 (0.3, 1 and 3 mg/kg IV once) and PBDdimer ADC disulfide prodrug 1B (1, 3, 6 and 10 mg/kg IV once). Tumorsand body weights of mice were measured 1-2 times a week throughout thestudy. Mice were promptly euthanized when body weight loss was >20% oftheir starting weight. All animals were euthanized before tumors reached3000 mm³ or showed signs of impending ulceration. Tumor volume wasmeasured in two dimensions (length and width) using calipers and thetumor volume was calculated using the formula: Tumor size(mm3)=0.5×(length×width×width).

The results are presented in FIGS. 23 to 26.

FIG. 23 shows efficacy of anti-CD22 ADCs in C.B-17 SCID mice withBJAB-luc human Burkitt's lymphoma. Non prodrug PBD dimer ADC control 2at the highest dose evaluated (0.2 mg/kg) only resulted in modest tumorgrowth delay. In contrast, PBD dimer ADC boronic acid prodrug 1A wasvery effective and drove tumor regression at the dose as low as 0.2mg/kg. The PBD dimer ADC boronic acid control 1 did not show any effecton the tumor growth. The PBD dimer ADC boronic acid prodrug 1A andnon-prodrug PBD dimer ADC control 3 conjugates had some anti-tumoractivity; however, the activity of anti-CD22 conjugates at matched doselevel was more superior.

FIG. 24 shows effect of the anti-CD22 ADCs (1) non prodrug PBD dimer ADCcontrol 2, (2) PBD dimer ADC boronic acid prodrug 1A and (3) PBD dimerADC boronic acid control 1 on the body weights of C.B-17 SCID mice withBJAB-luc human Burkitt's lymphoma. Administration of anti-CD22 ADCs waswell tolerated in animals with no body weight loss observed.

FIG. 25 shows efficacy of anti-Her2 ADCs in C.B-17 SCID-beige mice withKPL-4 human breast tumors. Non-prodrug PBD dimer ADC control 1demonstrated dose-dependent inhibition of tumor growth with tumorregression at 1 mg/kg or above. Similarly, PBD dimer ADC disulfideprodrug 1 also showed dose-dependent efficacy with tumor regression at 6mg/kg or above.

FIG. 26 shows effect of non-prodrug PBD dimer ADC control 1 and PBDdimer ADC disulfide prodrug 1 (anti-Her2 ADCs) on the body weights ofC.B-17 SCID-beige with KPL4 human breast tumors. The KPL4 xenograft is acachexia-inducing model, where animals lose about 5% of their initialbody weight in response to the tumor itself. Administration of anti-Her2ADC attenuated this tumor associated weight loss, and was well toleratedin animals.

Example 15: Toxicity of a DTD-Activated PBD Monomer and Dimer QuinoneProdrugs

The toxicity of PBD dimer diaphorase prodrug 1, PBD monomer diaphoraseprodrug 2, PBD dimer control 1 and PBD monomer control 1 were evaluatedagainst KPL-4 and WSU cell lines for cell viability. The KPL-4 cell lineis was a high DTD cell line characterized by NQO1 nRPKM of 839 and theWSU cell line was a low DTD cell line characterized by a NQO1 nRPKM of1.36. The IC₅₀ ratios are based on the prodrug IC₅₀ value relative tothe PBD dimer control.

The results are reported below in Table 10 and in FIGS. 17 to 20.

TABLE 10 IC₅₀ Potency Compound (nM) Ratio KPL-4 Cell line PBD dimerdiaphorase prodrug 1 35.48 17 PBD dimer control 1 2.09 PBD monomerdiaphorase prodrug 2 343.76 1 PBD monomer control 1 330.11 WSU Cell linePBD dimer diaphorase prodrug 1 8.56 43 PBD dimer control 1 0.2 PBDmonomer diaphorase prodrug 2 574.65 5 PBD monomer control 1 105.53

Example 16: Toxicity of a DTD-Activated PBD Monomer and Dimer QuinoneProdrugs

The toxicity of PBD dimer ADC diaphorase prodrugs 1A and 1B andnon-prodrug PBD dimer ADC control 5 were evaluated against KPL-4 andSK-BR-3 cell lines for cell viability. The KPL-4 cell line is was a highDTD cell line characterized by NQO1 nRPKM of 839 and the SK-BR03 cellline was a low DTD cell line characterized by a NQO1 nRPKM of 171. Theresults are reported below in FIGS. 21 and 22.

Example 17: Disulfide Cleavage and DNA Oligo Binding of PBD Analogs

The cleavage of various prodrug disulfide compounds of the presentdisclosure after 24 hour exposure to cysteine and glutathione (GSH) wasevaluated and the DNA binding of the PBD analogs were evaluated.

For disulfide cleavage determination, the compounds were incubated at 15μM with 0.2 mM cysteine or 4 mM GSH in 100 mM Tris buffer pH 7.0containing 5% methanol at 37° C. Aliquots were taken at specified timepoints and the samples were analyzed by LC/MS on Sciex TripleTOF 5600 ona Hypersil Gold C18 column (100×2.1, 1.9 μM, Thermo Scientific). Thecolumn was eluted by a gradient of buffer A (0.1% formic acid in 10 mMammonium acetate) to buffer B (0.1% formic acid in 10 mM ammoniumacetate in 90% acetonitrile), 5% B 0-0.5 min, 5-25% B 0.5-8 min, 25-75%B 8-13 min, and 75-95% B 13-13.5 min, 95% B 13.5-14.5 min, 95-5% B14.5-15 min at 0.4 mL/min. All products were separated and characterizedby LC/MS/MS in a positive ESI ion mode. All analytes had the protonatedmolecular MH+ as the major species with little source fragmentation.Full scan accurate mass peak areas were used to estimate relativeabundance of each component.

For DNA binding determination, the compounds were incubated at 100 μMwith 100 μM double strand DNA Oligos 1 and 2 for 1 hour in 10 mMBis-Tris, pH 7.1 at 37° C. The single strand DNA Oligos(5′-TATAGAATCTATA-3′ and 3′-ATATCTTAGATAT-5′) were synthesized atGenentech. The samples were analyzed by LC/MS/UV (210-450 nm) on SciexTripleTOF 5600 on a Hypersil Gold C18 column (100×2.1, 1.9 μM, ThermoScientific). The column was eluted at 0.4 mL/min by a gradient of bufferA (50 mM hexafluoro-isopropanol and 15 mM diisopropylethylamine) tobuffer B (50% A and 50% of 1:1 methanol:acetonitrile), 5% 0-0.5 min,5-25% B 0.5-25 min, 25-95% B 25-40 min, and to 95% B 40-42 min. The %remaining was an average of the starting DNA oligos remaining inincubations (n=2). The products were characterized by LC/MS in anegative ESI ion mode.

The results are reported in Table 11 below.

TABLE 11 % Remaining % Remaining % DNA oligo PBD Agent at 24 h by Cys at24 h by GSH remaining PBD monomer control 1 NA NA  35% PBD monomercontrol 2 100 100 — PBD monomer disulfide prodrug 1 17.9 0.5 — PBDmonomer disulfide prodrug 2 77.6 0.4 (0) — PBD monomer disulfide prodrug3 73.5 2.90 — PBD monomer disulfide prodrug 4 99.9 99.9 — PBD monomerdisulfide prodrug 5 90.9 65.9 — PBD monomer disulfide prodrug 6 85.4 2.1— PBD monomer disulfide prodrug 7 96.1 89.7 — PBD monomer disulfideprodrug 8 86.7 0.5 — PBD monomer disulfide prodrug 9 81.1 1 — PBDmonomer disulfide prodrug 10 99.4 88.5 — PBD monomer disulfide prodrug11 14.4 0 — PBD monomer disulfide prodrug 12 72.7 9.5 — PBD monomerdisulfide prodrug 13 99.9 98.8 — PBD monomer disulfide prodrug 14 68.1 3— PBD monomer disulfide prodrug 15 99.2 (90.8) 85.2 (32.5) — PBD monomerdisulfide prodrug 16 98.6 73.2 — PBD monomer disulfide prodrug 17 34.9 0— PBD monomer disulfide prodrug 18 75.3 18.7 — PBD monomer disulfideprodrug 19 4.4 0 — PBD monomer disulfide prodrug 20 0 (34% at 4 h) 0 —PBD monomer disulfide prodrug 21 74.7 0 — PBD monomer disulfide prodrug22 1.4 0 — PBD monomer disulfide prodrug 23 41.6 0 — PBD monomerdisulfide prodrug 24 0.1 0 — PBD monomer disulfide prodrug 25 0 0 — PBDmonomer disulfide prodrug 26 29.1 0 — PBD monomer disulfide prodrug 27NA 0 — PBD monomer disulfide prodrug 28 62.2 0 — PBD monomer disulfideprodrug 29 0 0 — PBD monomer disulfide prodrug 30 47.1 0 — PBD dimerdisulfide prodrug 1 — — >99.5 PBD dimer control 1 — — <1 PBD dimercontrol 2 — — 100%

Example 18: GSH Adduct Formation and Stability of Quinones

The stability of quinones and PBD monomer and dimer diaphorase prodrugswithin the scope of the present disclosure upon exposure to GSH andrelated GSH adduct formation were evaluated.

For degradation analysis, the compounds were incubated at 25 μM with 15mM GSH in 200 mM Tris buffer pH 7.0 containing 5% methanol at 37° C. for3 hours. The control incubations were conducted without GSH. The sampleswere analyzed by LC/MS on Sciex TripleTOF 5600 on a Hypersil Gold C18column (100×2.1, 1.9 μM, Thermo Scientific). The column was eluted by agradient of buffer A (0.1% formic acid in 10 mM ammonium acetate) tobuffer B (0.1% formic acid in 10 mM ammonium acetate in 90%acetonitrile), 5% B 0-0.5 min, 5-25% B 0.5-8 min, 25-75% B 8-13 min, and75-95% B 13-13.5 min, 95% B 13.5-14.5 min, 95-5% B 14.5-15 min at 0.4mL/min. All products were separated and characterized by LC/MS/MS in apositive ESI ion mode. Full scan accurate mass peak areas were used toestimate relative abundance of each component.

An in vitro DT diaphorase activated drug release assay was used tomeasure NADPH depletion. DT diaphorase activated release of Norfloxacinand Payload (PBD) was measured by the in vitro depletion of NADPH. Theassay method was modified from the absorbance measurement of NADPH atA340 (Osman et. al, Chemico-Biological Interactions 147 (2004) 99-108)due to the interference of compounds. The depletion of NADPH wasmeasured by monitoring the decrease of fluorescence intensity of NADPHat 480 nm after being excited at 340 nM. The materials and reagents wereas follows: (1) Bovine Serum Albumin (BSA): Sigma cat # A7030-50G (≥98%(agarose gel electrophoresis), lyophilized powder, essentially fattyacid free, essentially globulin free; (2) Assay buffer: 50 mMTris-HCl/0.007% BSA buffer (pH 7.4); (3) DT Diaphorase preparation:Dissolve lyophilized human DT Diaphorase (Sigma Cat #D1315, Lot #SLBJ9723V, MW=32, 253 U/mg, 1.6 mg protein/vial, One unit will reduce 1micromole of Cytochrome C per min in the presence of Menadione substrateat 37° C.) in 8.1 ml H2O as 50 U/0.2 mg/ml (6.25 uM protein), Storealiquots at −20° C. Prior to assay, 2.5 fold dilute the stock in assaybuffer as 5× working solution (20 U/mL, 2.5 uM, 0.08 ug/mL); (4)β-Nicotinamide adenine dinucleotide phosphate (NADPH, reduced disodiumsalt hydrate, Sigma, cat # N6505-5G, >=94% (HPLC): prepare 48 mM NADPHstock in assay buffer and store at −20 C. Prior to assay, dilute thestock to 1 mM as the 5× working solution; (5) DT Diaphorase specificInhibitor: prepare 40 mM stock of Dicumarol (Sigma, Cat # M1390-5G,MW=336.29) in 0.13 N NaOH, stored at 4° C. Dilute the stock to 250 uM asthe 5× working solution; (6) Compounds: dilute the Norfloxacin and PBDconjugated compounds in assay buffer to 250 uM as the 5× workingsolution; and (7) 384 well black plate with clear bottom. For the assayprocedure: (1) The reaction mix containing 0.5 uM DT-D, 50 uM compound,200 mM NADPH was set up in a 384 well plate at 50 ul/well. For thecontrol with DT Diaphorase inhibitor, Dicumarol was added in thereaction mix at final 50 uM for each compound. Reaction mix containingNADPH and compound only was also added as the baseline controls. The DTDiaphorase was added in the last step; and (2) The reaction mixes wereincubated at room temperature for 5, 30, 90 min. The fluorescenceintensity (RFU) of DNAPH was recorded on M1000 plate reader (Tecan) withexcitation at 340 nM and emission at 480 nM. For data analysis, data wasanalyzed and plotted using Prism GraphPad 6. The depletion of NADPH wascalculated by the formula below at reaction time point at 90 minwherein: % NADPH depletion=[RFU_(without inhibitor)−(RFU_(with inhibitor)/RFU_(without inhibitor))]*100.

The results are reported in Table 12 below where: “% Rem.” refers topercent remaining after 3 hours; “Degr” refers to degradation; “Pay.Rel.” refers to payload release (MRM Qunat) (% recovery at 90-minrelative to 0 hours) based on 2 pM of starting material; and “NADPHDep.” Refers to NADPH depletion at 90 minutes.

TABLE 12 GSH Pay. NADPH Quinone % Rem. Adduct Degr Rel. Dep.

90 yes — — 95.99

87 yes — — 74.83

92.5 yes — — None

100 — — — None

0 — yes — 96.92

63 — yes 1% 94/0

37.6 — yes 2% 53.23

82.1 — yes — 91.5

— — — 2% 85.2

50 — yes — 2.01

55.5 — yes — 26.1

100 — — — 17.4 PBD monomer diaphorase prodrug 1 — — — — >90% at 30 minPBD monomer diaphorase prodrug 2 — — — — >90% in <5 min PBD monomerdiaphorase prodrug 3 — — — — >90% at 90 min

Example 19: Preparation of PBD Monomer Disulfide Prodrugs Example 19General Scheme 1

The overall reaction scheme for general scheme 1 was as follows:

Example 19 General Scheme 1 Step 1

To a solution of compound 1 (1.13 kg, 4.59 mol, 1.00 Eq) in THF (10 L)at 0° C. was added LiBH₄ (99.90 g, 4.59 mol, 1.00 Eq) in two portions(almost no temperature charge during the adding of LiBH₄). Thesuspension was stirred at 0° C. for 1 h then at 10-20° C. for 18 h. Themixture was cooled to 0° C. and aq NH₄Cl (5 L) were added. The layerswere separated and the aqueous layer was extracted with EA (5 L×3). Thecombined organics were washed with brine. The organic layers were driedover Na₂SO₄, filtered and concentrated to afford compound 2 as a clearoil (1600 g, 7.36 mol, 80.22% yield).

Example 19 General Scheme 1 Step 2

To a 50-L flask was charged compound 2 (1.60 kg, 7.36 mol, 1.00 Eq), DCM(20 L) followed by the addition TEA (1.12 kg, 11.05 mol, 1.50 Eq) andacetyl chloride (635.54 g, 8.10 mol, 1.10 Eq) dropwise in turn withstirring at 0° C. After the addition, the resulting solution was stirredat 15-25° C. for 18 h, quenched by the addition of 5 L of water andextracted with 3×2L of DCM. The combined organic layers were dried overanhydrous sodium sulfate and concentrated under vacuum to affordcompound 3 as a colorless oil (2.46 kg, 9.49 mol, 128.90% yield).

Example 19 General Scheme 1 Step 3

To a 20 L 3-necked round-bottom flask was charged compound 3 (1.23 kg,4.75 mol, 1.00 Eq) in DCM (12 L) followed by the addition of PCC (1.54kg, 7.13 mol) in several batches at 15° C. The resulting solution wasstirred at 15-25° C. for 18 h. The solids were filtered off and thefiltrate was concentrated under vacuum. The residue was purified on asilica gel column eluting with ethyl acetate: petroleum ether (1:5) toafford compound 4 as a light yellow liquid (1.13 kg, 4.38 mol, 46.15%yield).

Example 19 General Scheme 1 Step 4

To a 10-L 3-necked round-bottom flask was chargedmethyl(triphenyl)phosphonium bromide (958.03 g, 2.68 mol), THF (2.5 L),followed by the addition of t-BuOH (300.94 g, 2.68 mol) in portions at0° C. over 2 h. To this was added a solution compound 4 (460.00 g, 1.79mol) in THF (2.5 L) dropwise with stirring at 0° C. The resultingsolution was stirred at −5˜0° C. for 20 min, quenched by the addition of500 mL of water and extracted with 3×500 mL of ethyl acetate. Thecombined organic layers were dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluting with ethyl acetate: petroleum ether (1:20) to affordcompound 5 as a light yellow liquid (275.00 g, 1.08 mol, 30.09%).

Example 19 General Scheme 1 Step 5

A mixture of compound 5 (330.00 g, 1.29 mol) in HCl (gas)/EtOAc (3 L,4M/L) was stirred at 0° C. 20 mins. Then the mixture was stirred at10-30° C. for 1 h. The mixture was concentrated in vacuum to affordcompound 6 as yellow solid (250.00 g, 1.30 mol, 101.12%), which is usedin next step without purification.

Example 19 General Scheme 1 Step 6

Into a 3000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was charged a solution of compound 7(354.42 g, 1.56 mol, 1.30 Eq) in THF (1.5 L), followed by the additionof SOCl₂ (1.71 kg, 14.33 mol, 11.94 Eq) dropwise with stirring. Theresulting solution was stirred at 20-30° C. for 4 h and thenconcentrated under vacuum. Into another 3000-mL 3-necked round-bottomflask purged and maintained with an inert atmosphere of nitrogen wascharged a solution of compound 6 (230.00 g, 1.20 mol, 1.00 Eq) in DCM(2.5 L). To this was added Et₃N (485.75 g, 4.80 mol, 4.00 Eq) dropwisewith stirring at −40° C., followed by the solution in the first flask at−40° C. The temperature was allowed to warm to 0° C. naturally, quenchedby the addition of 3000 mL of water/ice and extracted with 3×1000 mL ofdichloromethane. The combined organic layers were dried over anhydroussodium sulfate and concentrated under vacuum. The residue was purifiedon a silica gel column eluting with EtOAc:PE (1:3) to afford compound 8as a light brown oil (210.00 g), which is used in next step withoutpurification.

Example 19 General Scheme 1 Step 7

To a mixture of compound 8 (90.00 g, 247.02 mmol, 1.00 Eq) in THF (400mL), MeOH (100 mL), H2O (400 mL), was added NaOH (29.64 g, 741.05 mmol,3.00 Eq) in one portion at 0° C. The mixture was stirred at 20-30° C.for 18 h. The aqueous phase was extracted with EtOAc (300 mL×3). Thecombined organic phase was washed with saturated brine (100 mL), driedwith anhydrous Na₂SO₄, filtered and concentrated in vacuum to affordcompound 9 as yellow solid (90.26 g, crude), which was used for the nextstep without further purification.

Example 19 General Scheme 1 Step 8

In a 2000 mL three-necked round bottom flask equipped with a temperatureprobe, magnetic stirrers and a nitrogen inlet, TBDMSCl (126.62 g, 840.12mmol), imidazole (57.20 g, 840.12 mmol, 3.00 Eq) in DMF (1 L) wereadded. Then a solution of compound 9 (90.26 g, 280.04 mmol, 1.00 Eq) inDMF (1 L) was added to the mixture at 0° C. The resulting reactionmixture was stirred for 2 h at 25-30° C. The reaction mixture was pouredinto ice-water (1 L) and then extracted with DCM (200 mL×3). Thecombined organic phases were washed brine (100 mL), dried over Na₂SO₄and concentrated in vacuum to give the residue, to give compound 10 as ayellow oil (126.00 g), which was used for the next step without furtherpurification.

Example 19 General Scheme 1 Step 9

To a mixture of compound 10 (126.00 g, 288.61 mmol, 1.00 Eq) in AcOH (1L), was added Zn (188.72 g, 2.89 mol) in portions by maintaining thetemperature below 30° C. The mixture was stirred at 20-30° C. for 30min. The residue was poured into EtOAc (500 mL) and filtered. Thefiltrate was concentrated in vacuum. The residue was purified by silicagel chromatography (PE/EtOAc=10/1, 1/1) to afford 11 as yellow oil(58.00 g, 142.64 mmol, 49% yield). 1H NMR (400 MHz, CHLOROFORM-d) d ppm6.71 (s, 1H) 6.22 (s, 1H) 4.85-4.97 (m, 2H) 4.52 (br. s., 1H) 4.14-4.23(m, 1H) 3.99-4.13 (m, 1H) 3.82 (s, 3H) 3.77 (s, 3H) 3.59 (d, J=5.73 Hz,1H) 2.63-2.72 (m, 2H) 2.01-2.04 (m, 1H) 1.23 (t, J=7.06 Hz, 1H) 0.85 (s,9H)−0.06-0.06 (m, 5H).

Example 19 General Scheme 2 as Follows is a General Scheme for PreparingPBD Disulfide Prodrugs of the Present Disclosure

The asterisk in structure C, and elsewhere depicted in Example 19,represents a chiral center. In some aspects, R¹ recited in the abovescheme corresponds to R⁶¹ as described herein, R² recited in the abovescheme corresponds to R⁶² as described herein, and R³ recited in theabove scheme corresponds to R⁵⁰ as described herein.

Example 19A: Preparation of PBD Monomer Disulfide Prodrug 10

Example 19A General Procedure IA—Formation of Carbomate Method A

(2-amino-4,5-dimethoxy-phenyl)-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidin-1-yl]methanone(222 mg, 0.5460 mmol) was dissolved in 3 mL of DCM, then Et₃N was addedvia a pipette followed by triphosgene. After total of 20 min,2-(tert-butyldisulfanyl)-2-methyl-propan-1-ol (B, 1.05 equiv., 0.5733mmol, 100 mass %) in 2 mL of DCM (including 0.5 mL of rinsing) was addedfollowed by 10 uL of dibutyltin diacetate (20 μL, 0.07487 mmol). Afterabout 1.5 hr, another 35 uL (28 mg) of disulfide alcohol and 8 uL ofdibutyltin diacetate (2:57 pm) were added and the reaction was stirredovernight. The reaction was diluted with EtOAc, then washed with 1N HClsolution. The organics were washed with saturated sodium bicarbonate.The organic layer was dried over sodium sulfate and then concentrated.The residue was purified by flash chromatography (25 g silica gel,20%-30%-50% EtOPc/Hept) to give the desired carbomate as a colorless oil(227 mg, 67% yield).

Example 19A General Procedure II—Removal of TBS Group by HOAc

[2-(tert-butyldisulfanyl)-2-methyl-propyl]N-[2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidine-1-carbonyl]-4,5-dimethoxy-phenyl]carbamate(200 mg, 0.319 mmol) was dissolved in 3 mL of THF, then water (0.8 mL)and acetic acid (3.4 mL) were added at room temperature. After thereaction was done, sodium carbonate was added to neutralize acetic acid.The mixture was extracted with EtOAc three times. The combined EtOAcextract was dried over sodium sulfate, concentrated to provide the crudealcohol (233 mg), which was used in the next step without purification.

Example 19A General Procedure III—DMP Oxidation & Cyclization

To 2-(isopropyldisulfanyl)-2-methyl-propyl]N-[2-[(2S)-2-(hydroxymethyl)-4-methylene-pyrrolidine-1-carbonyl]-4,5-dimethoxy-phenyl]carbamate(99.2 mg, 0.199 mmol, 100 mass %) in DCM (3.5 mL, was added Dess-Martinperiodinane (86.1 mg, 0.203 mmol, 1.02 equiv.) at rt. The reactionmixture diluted with DCM, then washed with mixed saturated. NaHCO₃(about 3 mL) and 1 M sodium sulfite (about 3 mL), dried over sodiumsulfate, concentrated to give about 120 mg crude (oil), which waspurified by reverse-phase HPLC to give the desired carbomate (28.9 mg)along with recovered alcohol starting material (10.3 mg). LCMS: (5-95,AB, 5 min), RT=2.69 min, m/z=497 [M+1]+; 1H NMR (400 MHz, DMSO-d6) δ7.08 (s, 1H), 6.82 (s, 1H), 6.64 (d, J=6.0 Hz, 1H), 5.49-5.33 (m, 1H),5.13 (d, J=7.3 Hz, 2H), 4.21-4.05 (m, 2H), 4.04-3.91 (m, 1H), 3.81 (s,4H), 3.79-z3.72 (m, 1H), 3.46 (t, J=9.3 Hz, 1H), 3.31 (s, 3H), 2.99-2.76(m, 2H), 2.63-2.51 (m, 1H), 1.38-0.94 (m, 12H).

Example 19B: Preparation of PBD Monomer Disulfide Prodrug 4

The title compound was synthesized in the same manner as example 19Aexcept that General procedure IB below was used for the disulfideformation.

Example 19B General Procedure Ib—Formation of Carbomate Method B

(2-amino-4,5-dimethoxy-phenyl)-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidin-1-yl]methanone(121 mg, 0.2976 mmol) and N,N-diisopropylamine (3 equiv., 0.8928 mmol)was mixed in 3 mL of THF, then (4-nitrophenyl) carbonochloridate (75 mg,0.3720 mmol) was added at room temperature. The mixture was heated to75° C. After about 1 h 50 min,2-(isopropyldisulfanyl)-2-methyl-propan-1-ol (71 mg, 0.3938 mmol) in 1.5mL of THF was added. The mixture was stirred at 75° C. overnight, cooleddown to room temperature, diluted in EtOAc, washed with 1N HCl and thensat. sodium bicarb. The solution was concentrated and the resultingresidue was purified by silica gel chromatography (20% then 30% then 50%EtOAc/Hept) to give the disulfide (34 mg, 19% yield).

LCMS: (5-95, AB, 5 min), RT=2.78 min, m/z=511 [M+1]+; 1H NMR (400 MHz,DMSO-d6) δ 7.08 (s, 1H), 6.81 (s, 1H), 6.63 (d, J=5.9 Hz, 1H), 5.49-5.34(m, 1H), 5.13 (d, J=6.1 Hz, 2H), 4.19-3.91 (m, 3H), 3.81 (s, 3H),3.78-3.67 (m, 1H), 3.46 (t, J=9.2 Hz, 1H), 3.32 (s, 35H), 2.96-2.82 (m,1H), 2.61-2.53 (m, 1H), 1.18 (s, 9H), 1.04 (d, J=3.7 Hz, 6H).

Example 19C: Preparation of PBD Monomer Disulfide Prodrug 15

PBD monomer disulfide prodrug 15 was prepared according to the followingreaction scheme 3:

Triphosgene (116 mg, 0.39 mmol) was dissolved in 2 mL of DCM, then asolution of (2R)-2-[(5-nitro-2-pyridyl)disulfanyl]propan-1-ol (277 mg,1.125 mmol) and pyridine (0.14 mL, 1.761 mmol) in 2 mL of DCM was added.After 30 min, this solution was added to a solution of(2-amino-4,5-dimethoxy-phenyl)-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidin-1-yl]methanone (410 mg, 0.978 mmol, 97 mass %) and in 3mL of DCM (plus 0.5 mL of rinsing). After the reaction was done, themixture was diluted with EtOAc, washed with 1N aq. HCl and thensaturated sodium bicarbonate. The organics were dried over sodiumsulfate concentrated. The residue was purified by silica gel columnchromatography (25 g silica gel, 30% then 40% then 50% EtOAc/Hept) togive [(2R)-2-[(5-nitro-2-pyridyl)disulfanyl]propyl]N-[2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidine-1-carbonyl]-4,5-dimethoxy-phenyl]carbamate(480 mg, 72% yield).

To [(2R)-2-[(5-nitro-2-pyridyl)disulfanyl]propyl]N-[2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidine-1-carbonyl]-4,5-dimethoxy-phenyl]carbamate(45 mg, 0.066 mmol, 100 mass %) in DMF (0.5 mL) was addedpropane-2-thiol (40 mg, 0.53 mmol) via a syringe without any solvent.The mixture was heated to 59° C. in a sealed vial. After the reactionwas done, the mixture was concentrated under reduced pressure, azotropedwith toluene once. The resulting residue was purified by silica gelchromatography (40 g silica gel, 20%-35%-50% EtOAc/Hept) to give[(2R)-2-(isopropyldisulfanyl)propyl] N-[2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidine-1-carbonyl]-4,5-dimethoxy-phenyl]carbamateas a yellow oil (134 mg, 89% yield).

PBD monomer disulfide prodrug 15 was then prepared according to generalprocedures II and III of example 19A.

LCMS: (5-95, AB, 5 min), RT=2.58 min, m/z=483 [M+1]+; 1H NMR (400 MHz,DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (d, J=6.1 Hz, 1H), 5.38 (dd,J=9.7, 6.1 Hz, 1H), 5.20-5.07 (m, 2H), 4.20 (dd, J=11.1, 6.1 Hz, 1H),4.15-4.05 (m, 1H), 3.97 (d, J=15.4 Hz, 2H), 3.80 (s, 6H), 3.45 (t, J=9.3Hz, 1H), 3.10-2.82 (m, 3H), 2.58-2.53 (m, 1H), 1.18 (t, J=6.2 Hz, 7H),1.04 (d, J=6.9 Hz, 3H).

Example 19D: Preparation of PBD Monomer Disulfide Prodrug 13

PBD monomer disulfide prodrug 13 was prepared according to the method ofExample 19C. LCMS: (5-95, AB, 5 min), RT=2.70 min, m/z=497 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.81 (s, 1H), 6.65 (d, J=5.9 Hz,1H), 5.38 (dd, J=9.7, 5.9 Hz, 1H), 5.19-5.04 (m, 2H), 4.20 (dd, J=11.2,5.9 Hz, 1H), 4.10 (d, J=15.8 Hz, 1H), 4.04-3.91 (m, 2H), 3.80 (s, 6H),3.45 (t, J=9.3 Hz, 1H), 3.04-2.82 (m, 2H), 2.58-2.53 (m, 1H), 1.23 (s,9H), 1.03 (d, J=6.9 Hz, 3H).

Example 19E: Preparation of PBD Monomer Disulfide Prodrug 2

PBD monomer disulfide prodrug 2 was prepared according to the followingreaction scheme:

In some aspects, R³ corresponds to R⁵⁰ as described elsewhere herein.

Triphosgene (268 mg, 0.9046 mmol, 100 mass %) was dissolved in 3 mL ofDCM in a flask, then a solution of2-[(5-nitro-2-pyridyl)disulfanyl]ethanol (604 mg, 2.601 mmol) in 6 mL ofDCM was added followed by neat pyridine (1.8 equiv., 4.071 mmol, 100mass %). After 30 min, this solution was added to a solution of(2-amino-4,5-dimethoxy-phenyl)-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidin-1-yl]methanone(948 mg, 2.262 mmol) and in 5 mL of DCM (plus 0.8 mL of rinsing). Afterabout 20 min, the mixture was diluted with EtOAc, washed with 1N HCl andthen saturated sodium bicarbonate. The organics were dried over sodiumsulfate concentrated and columned (40 g silica gel, 25% then 45%EtOAc/Hept) to give the carbamate as a yellow fluffy solid (1.07 g, 71%yield).

2-[(5-nitro-2-pyridyl)disulfanyl]ethylN-[2-[(2S)-2-[[tert-butyl(dimethyl)silyl]oxymethyl]-4-methylene-pyrrolidine-1-carbonyl]-4,5-dimethoxy-phenyl]carbamate(1.067 g, 1.61 mmol) was dissolved in THF, then water (1.5 mL) was addedfollowed by acetic acid (8 mL) was added at rt. The mixture was heatedto 55° C. and stirred overnight. After the mixture was cooled to roomtemperature, sodium carbonate was added to neutralize acetic acid. Themixture was extracted with EtOAc three times. The combined EtOAc extractwas dried over sodium sulfate, concentrated to provide the crude alcohol(1.26 g), which was used in the next step without purification.

To the above alcohol (884 mg, 1.61 mmol) in DCM (16 mL) was addedDess-Martin periodinane (715 mg, 1.685 mmol) at room temperature. Afterabout 70 min, another 105 mg of D-M periodinane was added. After 2.5hrs, another 76 mg of D-M periodinane was added. As soon as the all thestarting material had consumed, the mixture was diluted with DCM, thenwashed with mixed saturated NaHCO₃ (about 6 mL) and 1 M sodium sulfite(about 6 mL), dried over sodium sulfate, concentrated under reducedpressure. The resulting residue was purified by silica gel columnchromatography (40 g silica gel, 50% then 80% then 100% EtOAc/Hept) togive 2-[(5-nitro-2-pyridyl)disulfanyl]ethyl(6S,6aR)-6,6a-dihydroxy-2,3-dimethoxy-8-methylene-11-oxo-7,9-dihydro-6H-pyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylateas a yellow solid (765 mg, 84% yield).

To 2-[(5-nitro-2-pyridyl)disulfanyl]ethyl(6aS)-6-hydroxy-2,3-dimethoxy-8-methylene-11-oxo-6,6a,7,9-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate(63 mg, 0.115 mmol) in 0.2 mL of DMSO was added propane-2-thiol (0.5mL,) via a syringe, then heated to 59° C. After the reaction was done,the mixture was cooled to room temperature, co-evaporated with EtOAc ina rotavap twice to remove as much of the thiol as possible. Theresulting residue was purified by reverse-phase HPLC to give2-sulfanylethyl(6aS)-6-hydroxy-2,3-dimethoxy-8-methylene-11-oxo-6,6a,7,9-tetrahydropyrrolo[2,1-c][1,4]benzodiazepine-5-carboxylate(38.6 mg).

LCMS: (5-95, AB, 5 min), RT=2.42 min, m/z=469 [M+1]+; 1H NMR (400 MHz,DMSO-d6) δ 7.06 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 5.37 (dd, J=9.6,5.4 Hz, 1H), 5.13 (d, J=7.0 Hz, 2H), 4.37 (dt, J=12.2, 6.3 Hz, 1H),4.14-3.93 (m, 4H), 3.80 (s, 6H), 3.45 (t, J=9.3 Hz, 1H), 3.01-2.83 (m,3H), 1.23-1.16 (m, 6H).

Example 19F: Preparation of PBD Monomer Disulfide Prodrug 16

PBD monomer disulfide prodrug 16 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=2.56 min, m/z=483 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.79 (s, 1H), 6.62 (s, 1H), 5.37(dd, J=9.7, 5.9 Hz, 1H), 5.16-5.09 (m, 2H), 4.35 (dt, J=12.2, 6.3 Hz,1H), 4.10 (d, J=16.0 Hz, 1H), 3.98 (d, J=16.0 Hz, 2H), 3.80 (s, 6H),3.44 (t, J=9.3 Hz, 1H), 2.88 (t, J=12.6 Hz, 3H), 2.54 (s, 1H), 1.25 (s,9H), 0.08 (s, 1H).

Example 19G: Preparation of PBD Monomer Disulfide Prodrug 29

PBD monomer disulfide prodrug 29 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=1.72 min, m/z=471 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.79 (d, J=4.9 Hz, 1H), 6.62 (s,1H), 5.37 (dd, J=9.7, 5.8 Hz, 1H), 5.13 (d, J=7.0 Hz, 2H), 4.88-4.80 (m,1H), 4.45-4.34 (m, 1H), 4.14-4.05 (m, 1H), 4.02-3.93 (m, 1H), 3.80 (s,6H), 3.79-3.78 (m, 1H), 3.60 (dq, J=14.2, 6.5 Hz, 2H), 3.49-3.40 (m,1H), 2.94-2.69 (m, 4H), 2.54 (s, 1H).

Example 19H: Preparation of PBD Monomer Disulfide Prodrug 28

PBD monomer disulfide prodrug 28 was prepared according to the method ofexample 19D. LCMS: (5-95, AB, 5 min), RT=2.68 min, m/z=495 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ7.06 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 5.37(dd, J=9.7, 5.8 Hz, 1H), 5.13 (d, J=6.8 Hz, 2H), 4.44-4.33 (m, 1H),4.15-4.05 (m, 1H), 4.02-3.93 (m, 1H), 3.81 (s, 6H), 3.44 (t, J=9.1 Hz,1H), 2.88 (d, J=7.8 Hz, 3H), 2.55 (dt, J=5.7, 2.5 Hz, 2H), 2.48-2.42 (m,1H), 1.89 (d, J=8.4 Hz, 2H), 1.64 (s, 2H), 1.53 (s, 4H).

Example 19I: Preparation of PBD Monomer Disulfide Prodrug 27

PBD monomer disulfide prodrug 27 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=1.83 min, m/z=499 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.80 (s, 1H), 5.37 (d, J=9.7 Hz,1H), 5.16-5.09 (m, 2H), 4.45-4.34 (m, 1H), 4.16-3.93 (m, 2H), 3.80 (s,6H), 3.44 (t, J=9.2 Hz, 1H), 3.05-2.79 (m, 5H), 2.65-2.50 (m, 1H), 2.46(s, 1H), 2.07 (s, 3H).

Example 19J: Preparation of PBD Monomer Disulfide Prodrug 7

PBD monomer disulfide prodrug 7 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=2.62 min, m/z=495 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.66-6.60 (m, 1H),5.41-5.36 (m, 1H), 5.16-5.09 (m, 2H), 4.30 (d, J=11.7 Hz, 1H), 4.15-4.06(m, 1H), 4.02-3.88 (m, 2H), 3.81 (s, 6H), 3.45 (t, J=9.3 Hz, 1H), 2.91(td, J=16.0, 8.6 Hz, 2H), 2.57-2.53 (m, 1H), 1.14 (dd, J=11.4, 6.6 Hz,6H), 0.96-0.81 (m, 4H).

Example 19K: Preparation of PBD Monomer Disulfide Prodrug 1

PBD monomer disulfide prodrug 1 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=2.20 min, m/z=455 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.80 (s, 1H), 6.62 (s, 1H), 5.37(dd, J=9.7, 5.1 Hz, 1H), 5.16-5.09 (m, 2H), 4.39 (dt, J=12.2, 6.3 Hz,1H), 4.15-3.93 (m, 3H), 3.80 (s, 6H), 3.45 (t, J=9.3 Hz, 1H), 2.88 (dd,J=16.1, 9.0 Hz, 3H), 2.65 (q, J=12.6, 6.8 Hz, 2H), 2.54 (d, J=2.3 Hz,1H), 1.23-1.14 (m, 3H).

Example 19L: Preparation of PBD Monomer Disulfide Prodrug 26

PBD monomer disulfide prodrug 26 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=2.08 min, m/z=497 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.65 (s, 1H), 5.38(d, J=9.7 Hz, 1H), 5.13 (d, J=6.6 Hz, 2H), 4.77 (s, 3H), 4.45-4.34 (m,1H), 4.26-4.06 (m, 3H), 4.02-3.93 (m, 1H), 3.81 (s, 6H), 3.45 (t, J=9.3Hz, 1H), 3.09 (s, 1H), 2.94-2.83 (m, 1H), 2.62-2.53 (m, 1H), 1.04 (d,J=6.9 Hz, 2H), -0.03-0.13 (m, 1H).

Example 19M: Preparation of PBD Monomer Disulfide Prodrug 25

PBD monomer disulfide prodrug 25 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=2.16 min, m/z=499 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (br s, 1H),5.38 (d, J=9.7 Hz, 1H), 5.14 (s, 2H), 4.2-4.1 (m, 2H), 3.99 (s, 1H),3.81 (s, 6H), 3.46 (s, 4H), 3.22 (s, 3H), 2.79 (s, 2H), 2.69-2.54 (m,3H), 1.05 (s, 2H).

Example 19N: Preparation of PBD Monomer Disulfide Prodrug 24

PBD monomer disulfide prodrug 24 was prepared according to the method ofexample 19E.

The thiol was synthesized according to the following scheme:

To a solution of tetrahydro-2H-pyran-4-carbaldehyde 14a (5 g, 43.8 mmol)in MTBE (50 mL) was added S₂Cl₂ (2.96 g, 21.9 mmol). The reactionmixture was stirred at 55° C. for 16 hours under nitrogen, and then thereaction mixture was cooled to ambient temperature, removed solventunder vacuum and purified by silica gel column chromatography(silica:200-300 mesh, PE/EtOAc=5/1) to give disulfide 14b (5 g, 58%) asa yellow oil. 1H NMR (300 MHz, CDCl3) δ 9.05 (s, 1H), 3.92-3.42 (m, 8H),2.22-1.58 (m, 8H).

To a solution of4,4′-disulfanediylbis(tetrahydro-2H-pyran-4-carbaldehyde) 7 (5 g, 12.82mmol) in THF (50 mL) was added LiAlH₄ (0.97 g, 25.64 mmol) in portions.After addition, the reaction mixture was stirred at ambient temperaturefor 2 hours, and then the reaction mixture was acidified with HCl (3 N)to PH=6, extracted with ethylacetate (30 mL×3), dried over Na₂SO₄,removed solvent and purified with silica gel column chromatography(silica:200-300 mesh, PE/EA=10/1) to give thiol 14c (2.02 g, 34%) asyellow oil. 1H NMR (300 MHz, CDCl3) δ 3.86-.383 (m, 4H), 3.53 (s, 2H),2.25 (s, 1H), 1.86-1.43 (m, 5H). GCMS (m/z) ES=148.

LCMS: (5-95, AB, 5 min), RT=2.24 min, m/z=525 [M+1]+; 1H NMR (400 MHz,DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.61 (s, 1H), 5.37 (d, J=9.6 Hz,1H), 5.13 (d, J=7.0 Hz, 2H), 4.23 (dd, J=11.1, 6.0 Hz, 1H), 4.10 (d,J=15.7 Hz, 1H), 4.02-3.83 (m, 4H), 3.81 (s, 6H), 3.45 (t, J=9.3 Hz, 1H),3.02 (dt, J=11.1, 4.1 Hz, 1H), 2.94-2.83 (m, 2H), 2.59-2.52 (m, 2H),1.95-1.76 (m, 2H), 1.55-1.37 (m, 2H), 1.27-1.21 (m, 1H), 1.05 (d, J=6.9Hz, 3H).

Example 190: Preparation of PBD Monomer Disulfide Prodrug

The above PBD monomer disulfide prodrug was prepared according to themethod of example 19E. LCMS: (5-95, AB, 5 min), RT=1.86 min, m/z=485[M+1]+; 1H NMR (400 MHz, DMSO-d6) δ 7.06 (s, 1H), 6.79 (s, 1H), 6.62 (s,1H), 5.37 (dd, J=9.7, 6.0 Hz, 1H), 5.13 (d, J=7.0 Hz, 2H), 4.88 (t,J=5.7 Hz, 1H), 4.38 (d, J=7.5 Hz, 1H), 4.16-3.95 (m, 2H), 3.80 (s, 6H),3.46 (d, J=11.3 Hz, 2H), 3.38-3.34 (m, 1H), 2.86 (s, 4H), 2.58-2.53 (m,1H), 1.19 (dd, J=12.6, 6.6 Hz, 3H).

Example 19P: Preparation of PBD Monomer Disulfide Prodrug 12

PBD monomer disulfide prodrug 12 was prepared according to the method ofexample 19E. LCMS: (5-95, AB, 5 min), RT=2.40 min, m/z=469 [M+1]+; 1HNMR (400 MHz, DMSO-d6) δ 7.07 (s, 1H), 6.82 (s, 1H), 6.63 (s, 1H), 5.38(dd, J=9.7, 5.9 Hz, 1H), 5.13 (d, J=6.7 Hz, 2H), 4.21 (dd, J=11.1, 6.2Hz, 1H), 4.10 (d, J=16.0 Hz, 1H), 3.97 (d, J=16.1 Hz, 2H), 3.81 (d,J=2.3 Hz, 6H), 3.45 (t, J=9.3 Hz, 1H), 3.05 (s, 1H), 2.94-2.83 (m, 1H),2.70-2.53 (m, 3H), 2.45 (p, J=1.9 Hz, 1H), 1.16 (t, J=7.2 Hz, 3H),1.08-1.02 (m, 2H).

Example 19Q: Preparation of PBD Monomer Disulfide Prodrug 3

PBD monomer disulfide prodrug 3 was prepared according to the method ofexample 19E.

The para-nitropyridine disulfide was synthesized according to thefollowing scheme:

To a solution of cyclobutanecarbaldehyde 17a (9.8 g, 120 mmol) in MTBE(80 mL) was added S₂Cl₂ (8.1 g, 60 mmol). The reaction mixture wasstirred at 55° C. for 16 hours under nitrogen. The reaction mixture wascooled to ambient temperature, remove solvent under vacuum and purifiedwith silica gel column chromatography (silica:200-300 mesh,PE/EtOAc=20/1) to give 1,1′-disulfanediyldicyclobutanecarbaldehyde (11.2g, 83%) as brown oil. 1H NMR (300 MHz, CDCl3) δ 9.28 (s, 2H), 3.06-1.20(m, 12H).

To a solution of 1,1′-disulfanediyldicyclobutanecarbaldehyde 5 (11.2 g,49 mmol) in THF (200 mL) was added LiAlH₄ (3.7 g, 97 mmol) in portions.After addition, the reaction mixture was stirred at ambient temperaturefor 2 hours. The reaction mixture was acidified with HCl(3 N) to pH=6,extracted with ethylacetate (200 mL×3), dried over Na₂SO₄, removesolvent and purified with silica gel column chromatography(silica:200-300 mesh, PE/EtOAc=10/1) to give thiol 17c (3.8 g, 33%) asyellow oil. 1H NMR (300 MHz, CDCl3) δ 3.64 (s, 2H), 2.35-2.09 (m, 5H),2.05-1.87 (m, 2H), 1.81 (s, 1H). GCMS (ES) m/z=+118.

A mixture of 17c (4.88 g, 41.35 mmol) and1,2-bis(5-nitropyridin-2-yl)disulfane (12.82 g, 41.35 mmol) in MeOH (100mL) was stirred at ambient temperature for 16 hours under nitrogen. Thesolution was concentrated under vacuum and the residue was purified bysilica gel column chromatography (silica:200-300 mesh, PE/EA=10/1) togive target compound 17d (2.01 g, 18%) as yellow solid. 1H NMR (400 MHz,DMSO) δ9.30 (s, 1H), 8.34 (dd, J=8.8, 2.6 Hz, 1H), 7.60 (d, J=8.8 Hz,1H), 3.57 (s, 2H), 3.41 (s, 1H), 2.27-2.14 (m, 5H), 2.08-1.93 (m, 1H);LCMS (ES) m/z=+273 (M+1).

LCMS: (5-95, AB, 5 min), RT=2.61 min, m/z=495 [M+1]+; 1H NMR (400 MHz,DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.64 (d, J=6.0 Hz, 1H), 5.45-5.36(m, 1H), 5.13 (d, J=7.2 Hz, 2H), 4.30 (d, J=11.5 Hz, 1H), 4.10 (d,J=15.8 Hz, 1H), 3.97 (d, J=13.7 Hz, 2H), 3.80 (s, 6H), 3.46 (td, J=9.5,1.8 Hz, 1H), 2.89 (dd, J=15.8, 9.2 Hz, 1H), 2.62-2.52 (m, 2H), 1.91 (s,6H), 1.63 (s, 1H), 1.13 (t, J=7.5 Hz, 3H).

Example 19R: Preparation of PBD Monomer Disulfide Prodrug 5

PBD monomer disulfide prodrug 5 was prepared according to the method ofexample 19E.

The para-nitropyridine disulfide was synthesized according to thefollowing scheme:

To a solution of cyclopentanecarbaldehyde 18a (9.0 g, 92 mmol) in MTBE(30 mL) was added S₂Cl₂ (7.4 g, 55 mmol). The reaction mixture wasstirred at 55° C. for 16 hours under nitrogen. The reaction mixture wascooled to ambient temperature, removed solvent under vacuum and purifiedwith silica gel column chromatography (silica:200-300 mesh,PE/EtOAc=80/1) to give 1,1′-disulfanediyldicyclopentanecarbaldehyde 18b(5.5 g, 46%) as brown oil. 1H NMR (300 MHz, CDCl3) δ 9.23 (s, 2H),2.41-1.51 (m, 16H).

To a solution of 1,1′-disulfanediyldicyclopentanecarbaldehyde 18b (8.5g, 32.9 mmol) in THF (60 mL) was added LiAlH₄ (2.5 g, 65.8 mmol) inportions. After addition, the reaction mixture was stirred at ambienttemperature for 2 hours, and then the solution was acidified with HCl (3N) to pH=6, extracted with ethylacetate (150 mL×2), dried over Na₂SO₄,removed solvent and purified by silica gel column chromatography(silica:200-300 mesh, PE/EtOAc=20/1) to give 18c (5.5 g, 63%) as yellowoil. 1H NMR (300 MHz, CDCl3) δ 3.51 (s, 2H), 2.04 (s, 1H), 1.85-1.67 (m,7H), 1.64 (s, 1H). GCMS (ES) m/z=+132.

A mixture of 18c (3.5 g, 26.5 mmol) and1,2-bis(5-nitropyridin-2-yl)disulfane 3 (12.3 g, 39.8 mmol) in MeOH (50mL) was stirred at ambient temperature for 16 hours under nitrogen.After the reaction was completed, the solution was concentrated undervacuum. The residue was purified with silica gel column chromatography(silica:200-300 mesh, PE/EtOAc=10/1) to give target compound 18d (1.9 g,25%) as a yellow solid. 1H NMR (400 MHz, DMSO): δ 9.24-9.20 (m, 1H),8.58 (dd, J=8.9, 2.7 Hz, 1H), 8.17 (dd, J=8.9, 0.5 Hz, 1H), 5.18 (t,J=5.5 Hz, 1H), 3.40 (d, J=5.5 Hz, 2H), 1.63-1.82 (m, 8H); LCMS (ES)m/z=+287 (M+1).

LCMS: (5-95, AB, 5 min), RT=2.48 min, m/z=509 [M+1]+; 1H NMR (400 MHz,DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H), 6.73-6.60 (m, 1H), 5.40 (d, J=7.7Hz, 1H), 5.13 (d, J=7.2 Hz, 2H), 4.22 (d, J=11.0 Hz, 1H), 4.10 (d,J=15.9 Hz, 1H), 4.01-3.84 (m, 2H), 3.81 (s, 6H), 3.46 (t, J=9.2 Hz, 1H),2.88 (dd, J=15.9, 9.3 Hz, 1H), 2.55 (dd, J=4.1, 2.3 Hz, 2H), 1.81-1.39(m, 10H), 1.12 (t, J=7.3 Hz, 3H).

Example 19S: Preparation of PBD Monomer Disulfide Prodrugs 11 and 30

PBD monomer disulfide prodrugs 11 and 30 correspond to the abovestructure and are are diastereomers having a different configuration atone or more of the chiral centers designated with the asterisk. Thecompounds were prepared according to the method of example 19E.

The para-nitropyridine disulfide was synthesized according to thefollowing scheme using the procedure described in Example 19N.

PBD monomer disulfide prodrug 11 LCMS: (5-95, AB, 5 min), RT=1.95 min,m/z=511 [M+1]+; 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.83 (s, 1H),6.64 (s, 1H), 5.39 (d, J=9.7 Hz, 1H), 5.13 (d, J=7.1 Hz, 2H), 4.34 (d,J=11.5 Hz, 1H), 4.10 (d, J=16.0 Hz, 1H), 3.98 (t, J=15.3 Hz, 2H), 3.81(d, J=2.4 Hz, 6H), 3.78-3.72 (m, 1H), 3.63 (s, 1H), 3.54 (s, 2H), 3.46(t, J=9.2 Hz, 1H), 2.89 (dd, J=15.7, 9.4 Hz, 1H), 2.62-2.50 (m, 3H),1.83 (d, J=37.2 Hz, 2H), 1.12 (t, J=7.3 Hz, 3H).

PBD monomer disulfide prodrug 30 LCMS: (5-95, AB, 5 min), RT=1.99 min,m/z=511 [M+1]+; 1H NMR (400 MHz, DMSO-d6) δ 7.08 (s, 1H), 6.82 (s, 1H),6.66 (s, 1H), 5.48-5.34 (m, 1H), 5.21-5.07 (m, 2H), 4.33 (d, J=11.5 Hz,1H), 4.18-3.91 (m, 3H), 3.81 (s, 6H), 3.79-3.70 (m, OH), 3.48 (d, J=17.5Hz, 2H), 2.97-2.82 (m, 1H), 2.55 (dd, J=4.2, 2.2 Hz, 2H), 2.47-2.29 (m,1H), 1.85 (d, J=13.2 Hz, 2H), 1.13 (t, J=7.4 Hz, 3H).

Example 20: Preparation of PBD Dimer Disulfide Prodrugs Example 20A:Preparation of PBD Dimer Disulfide Prodrug 4

PBD dimer disulfide prodrug 4 was prepared according to the followingreaction scheme:

Each asterisk in the above structure, and elsewhere depicted in Example20, represents a chiral center.

To a solution of triphosgene (83.2 mg, 0.280 mmol) in DCM (2.0 mL) wasadded a solution of compound A2 and pyridine in DCM (3.0 mL) at 0° C.The mixture was stirred at 20° C. for 30 min and was concentrated invacuo. It was dissolved in DCM (5.0 mL) and added dropwise to a solutionof pyridine (18.5 mg, 0.234 mmol) and compound A1 (124 mg, 0.516 mmol)at 20° C. After the reaction mixture was stirred at 20° C. for 2 h, itwas concentrated in vacuo and purified by column chromatography (0-50%EtOAc in petroleum ether) to give compound A3 as a yellow solid (150 mg,54%). LCMS (5-95, AB, 1.5 min): RT=1.419 min, m/z=1261.4 [M+1]+.

A solution of compound A3 (150 mg, 0.119 mmol) in THF (4.0 mL), H₂O (4.0mL) and HOAc (6.0 mL) was stirred at 10° C. for 8 h. The mixture wasdiluted with EtOAc (15 mL) and washed with H₂O (10 mL), aqueous NaHCO₃(10 mL), and brine (10 mL). The organic layer was dried over Na₂SO₄,filtered, concentrated and purified by prep-TLC (5% CH₃OH in DCM) togive compound A4 (75 mg, 60.4%) as a colorless oil. LCMS (5-95, AB, 1.5min): RT=0.941 min, m/z=1033.3 [M+1]+.

A mixture of compound A4 (44 mg, 0.04 mmol) and DMP (54 mg, 0.13 mmol)in DCM (15 mL) was stirred at 13° C. for 16 h. The reaction mixture wasconcentrated in vacuo and purified by prep-TLC (5.6% MeOH in DCMRf=0.5), followed by prep-HPLC (10 mM, NH₄HCO₃-ACN) to give PBD dimerdisulfide prodrug 4 (15 mg, 34%) as a white solid. LCMS (5-95, AB, 1.5min): RT=0.941 min, m/z=1051.2 [M+23]+; 1H NMR (400 MHz, CDCl3) δ 7.19(s, 2H), 6.73 (s, 2H), 5.59-5.57 (m, 2H), 5.12 (s, 4H), 4.43 (d, J=10.8Hz, 2H), 4.26-4.15 (m, 1H), 4.11 (s, 1H), 4.02-3.98 (m, 3H), 3.96 (s,4H), 3.88 (s, 6H), 3.80-3.65 (m, 2H), 3.62 (m, 2H), 2.90-2.80 (m, 2H),2.72-2.60 (m, 6H), 2.18 (br, 2H), 2.02-1.92 (br, 13H), 1.70-1.63 (m,3H), 1.22-1.19 (m, 6H).

Example 20B: Preparation of PBD Dimer Disulfide Prodrug 1

PBD dimer disulfide prodrug 1 was prepared according to the followingreaction scheme:

To a solution of triphosgene (42.02 mg, 0.140 mmol) in DCM (4.0 mL)added the solution of compound A1 (300.0 mg, 0.310 mmol) andtriethylamine (63.68 mg, 0.630 mmol) in DCM (4 mL) dropwise. After themixture stirred at 0° C. for 30 min, a solution of compound A2 (112.16mg, 0.630 mmol) and triethylamine (127 mg, 1.26 mmol) in DCM (2.0 mL)was added and the mixture was stirred at 18° C. for 18 h. The mixturewas partitioned between water (20.0 mL) and DCM (40.0 mL), and theorganic layer was washed with water (20.0 mL), brine (20.0 mL), andconcentrated. It was purified on column chromatography (EtOAc:petroleumether 1:2) to afford compound A3 (240 mg, 65%) as a yellow oil. LCMS(5-95, AB, 1.5 min): RT=1.296 min, m/z=1157.4 [M+1]+.

To a solution of compound A3 (240.0 mg, 0.210 mmol) in THF (1.5 mL) wasadded a mixture of HOAc/H2O (4.0 mL, 3/1) dropwise. The mixture wasstirred at 8° C. for 18 h. The pH was adjusted to 8 with a NaHCO₃solution, and it was extracted with EtOAc (3×50 mL). The combinedorganic layer was dried over Na₂SO₄, filtered and concentrated. It waspurified by column chromatography (DCM:MeOH=20:1) to give compound A4(130 mg, 68%) as a yellow oil. LCMS (5-95, AB, 1.5 min): RT=0.868 min,m/z=929.3 [M+1]+.

To a solution of compound A4 (60.0 mg, 0.0600 mmol) in DCM (6.0 mL)added DMP (95.83 mg, 0.230 mmol), and the mixture stirred at 18° C. for1.0 h. The mixture was filtered, washed with aqueous Na₂SO₃ (20.0 mL),brine (20.0 mL) and water (20.0 mL). The organic layer was dried overNa₂SO₄, concentrated, and purified by prep-TLC (7% MeOH in DCM) to givethe compound A5 (30 mg, 49%) as a white solid. LCMS (5-95, AB, 1.5 min):RT=0.804 min, m/z=925.3 [M+1]+.

TFA (1.0 mL) was added dropwise to compound A5 (30.0 mg, 0.030 mmol) at0° C. After it was stirred for 20 min, the mixture was added to a sat.NaHCO₃ solution (40.0 mL) dropwise at 0° C., and extracted with DCM(3×15 mL). The combined organic layer was dried over Na₂SO₄,concentrated, and purified by prep-TLC (18% MeOH in DCM, Rf=0.6) toafford PBD dimer disulfide prodrug 1 (5.8 mg, 22%) as a white solid.LCMS (5-95, AB, 1.5 min): RT=0.890 min, m/z=807.2 [M+1]+.

Example 20C: Preparation of PBD Dimer Disulfide Prodrug 2

PBD dimer disulfide prodrug 2 was prepared according to the followingreaction scheme:

To a solution of triphosgene (89.43 mg, 0.300 mmol) and 4 Å molecularsieves (50 mg) in DCM (5.0 mL) was added a solution of compound A2(165.0 mg, 0.710 mmol) and pyridine (168.58 mg, 2.13 mmol) in DCM (5.0mL). The mixture was stirred at 0° C. for 30 min. The resulting mixturewas added dropwise to a solution of compound A1 (745 mg, 0.780 mmol),pyridine (169 mg, 2.13 mmol) and 4 Å MS in DCM (5.0 mL). It was stirredat 0° C. for 30 min, and washed with water (5.0 mL). Organic phase wasdried, concentrated and purified by flash column chromatography (5% MeOHin DCM) to give the product A3 (698 mg, 81%) as a yellow oil. LCMS(5-95, AB, 1.5 min): RT=1.187 min, m/z=606.5 [M/2+1]+.

To a solution of compound A3 (698.0 mg, 0.580 mmol) in DCM (10.0 mL) wasadded 2-propanethiol (439 mg, 5.76 mmol). After the mixture was stirredat 20° C. for 1 h, MnO₂ (100 mg) was added and stirred for 5 min, andfiltered. The filtrate was concentrated and purified by prep-TLC (50%EtOAc in petroleum ether) to give compound A5 (620 mg, 95%) as a yellowsolid. LCMS (5-95, AB, 1.5 min): RT=1.221 min, m/z=1131.4 [M+1]+.

To a solution of compound A5 (620.0 mg, 0.550 mmol) in THF (6.0 mL) andwater (6.0 mL) was added HOAc (3.29 g, 54.8 mmol). The mixture wasstirred at 40° C. for 16 h and concentrated. It was purified by columnchromatography (10% MeOH in DCM) to afford compound A6 (208 mg, 42%) asyellow oil. LCMS (5-95, AB, 1.5 min): RT=0.854 min, m/z=903.3 [M+1]+.

To a solution of compound A6 (208.0 mg, 0.230 mmol) in DCM (8.0 mL) wasadded 4 Å molecular sieves, DMP (224.7 mg, 0.530 mmol). The mixture wasstirred at 20° C. for 2 h and was quenched with saturated NaHCO₃ andNa₂S203 solution (2.0 mL/2.0 mL). After it was stirred for 5 min, DCM(5.0 mL) was added and separated. DCM phase was washed with water (2×5mL). It was dried, concentrated and purified by prep-TLC (5% MeOH inDCM, Rf=0.2) to afford compound A7 (121 mg, 58%) as a light yellow foam.LCMS (5-95, AB, 1.5 min): RT=0.783 min, m/z=781.3 [M−100+1]+.

TFA (1.0 mL, 13.5 mmol) was added to compound A7 (121.0 mg, 0.130 mmol)at 0° C. After the mixture was stirred for 10 min, it was added to acold saturated NaHCO₃ solution (20 mL) and extracted with DCM (3×10 mL).The combined organic layer was concentrated and purified by prep-TLC(10% MeOH in DCM, Rf=0.2) followed by prep-HPLC (ACN, acetonitrile:42˜62%, 0.225% FA) to afford PBD dimer disulfide prodrug 2 (7.2 mg,7.0%). LCMS (5-95, AB, 1.5 min): RT=0.868 min, m/z=781.3 [M+1]+.

Example 20D: Preparation of PBD Dimer Disulfide Prodrug 3

PBD dimer disulfide prodrug 3 was prepared according to the followingreaction scheme:

A solution of triphosgene (65.37 mg, 0.220 mmol) in DCM (2.0 mL) wasadded a mixture of compound A1 (420.0 mg, 0.440 mmol) and triethylamine(89.16 mg, 0.880 mmol) in DCM (3.0 mL) at 0° C. under N₂. After themixture was stirred at 21° C. for 30 min, it was concentrated, and DCM(18 mL) was added. A solution of A2 (75.0 mg, 0.390 mmol) andtriethylamine (78.91 mg, 0.780 mmol) in DCM (2.0 mL) was added at 0° C.under N₂. After the reaction mixture was stirred at 20° C. for 1 h, itwas concentrated in vacuo and purified by column chromatography (0-50%EtOAC in petroleum ether) to give compound A3 (350 mg, 74%) as a yellowsolid. LCMS (5-95, AB, 1.5 min): RT=1.379 min, m/z=1171.5 [M+1]+.

To a solution of compound A3 (415.2 mg, 0.350 mmol) in THF (30 mL) andwater (15 mL) was added HOAc (2.02 mL, 35.2 mmol). The reaction solutionwas stirred at 20° C. for 12 h. It was concentrated in vacuo and dilutedwith EtOAc (200 mL), washed with H₂O (2×100 mL), then aq. NaHCO₃solution (2×60 mL). The EtOAc layer was dried over Na₂SO₄, filtered, andconcentrated. It was purified by column chromatography (0-10% MeOH inDCM) to afford compound A4 (320 mg, 87.1%) as a yellow solid. LCMS(5-95, AB, 1.5 min): RT=1.080 min, m/z=943.5 [M+1]+.

To a solution of compound A4 (170.0 mg, 0.180 mmol) in DCM (20 mL) wasadded DMP (229.3 mg, 0.540 mmol). After the reaction mixture was stirredat 18° C. for 1 h, it was diluted with H₂O (20 mL), and aq. Na₂SO₃solution (20 mL), and aq. NaHCO₃ solution (20 mL) were added. Themixture was extracted with EtOAc (3×60 mL). The combined organic layerswere dried over Na₂SO₄, filtered, concentrated, and purified by columnchromatography (0-5% MeOH in DCM) to give compound A5 (150 mg, 75%) as awhite solid. LCMS (5-95, AB, 1.5 min): RT=0.814 min, m/z=961.5 [M+23]+.

A solution of compound A5 (75.0 mg, 0.080 mmol) in TFA (9.5 mL) andwater (0.50 mL) was stirred at 14° C. for 1 h. The reaction mixture waspoured into cold saturated NaHCO₃ (100 mL) and extracted with DCM (2×100mL). The combined organic layer was dried, concentrated, and purified byprep-TLC (4% MeOH in DCM Rf=0.5) followed by prep-HPLC (Waters XbridgePrep OBD C18 150*30 5 u, Condition:0.225% FA-CAN) to give PBD dimerdisulfide prodrug 3 (9.5 mg, 14%) as a white solid. LCMS (5-95, AB, 1.5min): RT=0.956 min, m/z=821.3 [M+23]+.

Example 20E: Preparation of PBD Dimer Control 1

PBD dimer control 1 was prepared according to the following reactionscheme:

A solution of compound A1 (1.80 g, 1.71 mmol) in HOAc/THF/H₂O (9.0mL/4.5 mL/3.0 mL) was stirred at rt for 48 h. The solution was dilutedwith EtOAc (150 mL), washed with H₂O (4×40 mL), aq. NaHCO₃ (4×40 mL),and H₂O (40 mL). The EtOAc layer was dried over Na₂SO₄, filtered, andconcentrated to afford compound A2 (1.38 g, 91%) as an oil.

To a stirred solution of compound A2 (800 mg, 0.99 mmol) in DCM (20 mL)was added DMP (1.26 g, 2.97 mmol) at 0° C. The reaction mixture wasstirred at rt for 3 h. It was diluted with EtOAc (100 mL), and quenchedwith aq. Na₂SO₃ solution (30 mL) at 0° C. The organic layer was washedwith H₂O (3×30 mL), aq. NaHCO₃ solution (30 mL), and H₂O (30 mL). It wasdried over Na₂SO₄, filtered, concentrated, and purified by prep-TLC(DCM/MeOH=15:1) to afford compound A3 (400 mg, 50.0%) as colorlesssolid. LCMS (ESI, 5-95AB/1.5 min): RT=0.767 min, [M+Na]+=843.4.

A solution of compound A3 (300 mg, 0.36 mmol) in 95% TFA/H₂O (4.0 mL)was stirred at 0° C. for 2 h. Then the solution was added dropwise intoa saturated NaHCO₃ solution (120 mL) at 0° C. The mixture was extractedwith DCM (3×20 mL). The combined organic layer was dried over Na₂SO₄,filtered, dried, concentrated and purified by prep-HPLC to afford PBDdimer control 1 (70 mg, 33%) as a white solid. LCMS (ESI, 5-95AB/1.5min): RT=0.767 min, [M+Na]+=843.4 1H NMR (400 MHz, CDCl3) δ ppm 7.69 (d,J=4.80 Hz, 2H), 7.50 (s, 2H), 6.81 (s, 2H), 5.19 (d, J=10.80 Hz, 4H),4.29 (s, 5H), 4.02-4.19 (m, 4H), 3.94 (s, 6H), 3.83-3.92 (m, 3H),3.09-3.16 (m, 2H), 2.90-2.99 (m, 2H), 1.98-1.94 (m, 4H), 1.66-1.70 (m,2H).

Example 20F: Preparation of PBD Dimer Control 2

PBD dimer control 2 was prepared according to the following reactionscheme:

A solution of compound A1 (1.80 g, 1.71 mmol) in HOAc/THF/H₂O (9.0mL/4.5 mL/3.0 mL) was stirred at rt for 48 h. It was diluted with EtOAc(150 mL), washed with H₂O (4×40 mL), aq. NaHCO₃ (2×40 mL), and H₂O (40mL). The EtOAc layer was dried over Na₂SO₄, filtered, and concentratedto afford compound 2 (1.38 g, 91%) as an oil.

To a stirred solution of compound A2 (800 mg, 0.99 mmol) in DCM (20 mL)was added DMP (1.26 g, 2.97 mmol) at 0° C., and the reaction mixture wasstirred at rt for 3 h. Then the mixture was diluted with EtOAc (100 mL),and quenched with aq. Na₂SO₃ solution (30 mL) at 0° C. The organic layerwas washed with H₂O (3×30 mL), saturated NaHCO₃ solution (30 mL), andH₂O (30 mL), dried over Na₂SO₄, filtered, and concentrated. The residuewas purified by prep-TLC (DCM/MeOH=15:1) to afford compound A3 (400 mg,50.0%) as a colorless solid. LCMS (ESI, 5-95AB/1.5 min): RT=0.867 min,[M+Na]+=843.4. 1H NMR (400 MHz, CDCl3) δ 7.20 (s, 2H), 6.61 (s, 2H),5.49 (d, J=9.2 Hz, 2H), 5.14 (d, J=4.8 Hz, 4H), 4.32-4.14 (m, 5H),4.07-3.99 (m, 5H), 3.90 (s, 6H), 3.62 (t, J=9.2 Hz, 2H), 2.96-2.89 (m,2H), 2.73-2.69 (m, 2H), 1.98-1.94 (m, 4H), 1.69-1.67 (m, 2H), 1.37 (s,18H).

A solution of compound A3 (120 mg, 0.147 mmol) in 95% TFA/H2O (2.0 mL)was stirred at 0° C. for 2 h. Then the solution was added dropwise to asaturated NaHCO₃ solution (120 mL) at 0° C. The mixture was extractedwith DCM (3×20 mL). The combined organic layer was dried over Na₂SO₄,and concentrated to afford compound 4 (86 mg, 100%) as crude product.LCMS (ESI, 5-95AB/1.5 min): RT=0.764 min, [M+H]+=585.3.

To a solution of compound A4 (86 mg, 0.147 mmol) in anhydrous DCM/MeOH(5.0 mL/2.5 mL) was added NaBH₃CN (92 mg, 1.47 mmol). The reactionmixture was stirred at rt overnight. It was concentrated and the residuewas diluted with saturated NaHCO₃ (20 mL), and extracted with DCM (3×20mL). The combined DCM layer was dried over Na₂SO₄, filtered, andconcentrated. The residue was purified by prep-HPLC (FA) to afford PBDdimer control 2 (15.6 mg, 18.1%) as white solid. LCMS (ESI, 5-95AB/1.5min): RT=0.808 min, [M+H]+=589.3. 1H NMR (400 MHz, CDCl3) δ 7.58 (s,2H), 6.05 (s, 2H), 5.06 (d, J=11.6 Hz, 4H), 4.42-4.27 (m, 6H), 3.98 (t,J=6.8 Hz, 6H), 3.84 (s, 6H), 3.55 (d, J=12.0 Hz, 2H), 3.35-3.30 (dd,J=12.4, 9.6 Hz, 2H), 2.93-2.87 (m, 2H), 2.46-2.42 (m, 2H), 1.94-1.89 (m,4H), 1.62-1.60 (m, 2H).

Example 21: Preparation of PBD Dimer Disulfide Prodrugs Comprising aLinker for Conjugation to an Antibody Example 21A: Preparation of PBDDimer Disulfide Prodrug 4 Comprising a Linker

PBD dimer disulfide prodrug 4 comprising a linker was prepared accordingto the following reaction scheme:

Synthesis of A2

Each asterisk in the above structure, and elsewhere depicted in Example21, represents a chiral center.

To the mixture of A6 (400.0 mg, 1.47 mmol) in DCM (10 mL) was addedethanethiol A7 (2.74 g, 44.1 mmol). The reaction mixture was stirred at40° C. for 30 h. The mixture was treated with MnO₂ (0.20 g) for 5 min,and filtered. The filtrates were concentrated and the residue waspurified by prep-TLC (100% DCM, Rf=0.5) to give compound A2 (110 mg,42%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 3.74 (s, 2H),2.75-2.70 (m, 2H), 2.13-1.87 (m, 6H), 1.84 (s, 1H), 1.30 (t, J=7.6 Hz,3H).

To a solution of triphosgene (82.4 mg, 0.28 mmol) in DCM (4.0 mL) wasadded a solution of A2 (110.0 mg, 0.620 mmol) and pyridine (146.4 mg,1.85 mmol) in DCM (4.0 mL) dropwise. The mixture was stirred at 15° C.for 30 min and was concentrated. It was dissolved in DCM (5.0 mL) andadded dropwise to a solution of A1 (1.05 g, 1.23 mmol) and pyridine(145.87 mg, 1.84 mmol) in DCM (15.0 mL) at 0° C. After the mixture wasstirred at 15° C. for 2 h, it was concentrated and the residue waspurified by column chromatography (0-50% EtOAc in petroleum ether) toafford A3 (310 mg, 45.8%) as a yellow oil. LCMS (5-95, AB, 1.5 min):RT=1.151 min, m/z=1057.4 [M+1]+.

To the solution of triphosgene (26.5 mg, 0.090 mmol) in DCM (5.0 mL) wasadded a solution of A3 (210 mg, 0.200 mmol) and triethylamine (60.28 mg,0.60 mmol) in DCM (5.0 mL) at 0° C. The reaction mixture was stirred at15° C. for 30 min. To the above mixture was added a solution ofMC—VC-PAB (166.0 mg, 0.290 mmol) and triethylamine (59.0 mg, 0.58 mmol)in DMSO (3.0 mL) dropwise. The reaction mixture was stirred at 40° C.for 2 h. The mixture was diluted with DCM (30 mL) and washed with water(3×10 mL). The combined organic layer was dried, concentrated, andpurified by column chromatography (0-10% MeOH in DCM) to afford A4 (160mg, 48.4%) as a yellow oil. LCMS (5-95, AB, 1.5 min): RT=1.271 min,m/z=828.6 [M/2+1]+.

To the mixture of A4 (160.0 mg, 0.100 mmol) in THF (3.0 mL) and water(3.0 mL) was added acetic acid (4.5 mL). The reaction mixture wasstirred at 15° C. for 15 h. The mixture was diluted with EtOAc (30 mL),washed with water (2×10 mL), saturated NaHCO₃ (2×10 mL) and brine (10mL). The organic phase was dried over Na₂SO₄ and concentrated to givethe crude A5 (120 mg, 82.7%) as yellow oil, which was used in the nextstep without further purification. LCMS (5-95, AB, 1.5 min): RT=0.801min, m/z=714.6 [M/2+1]+.

To the mixture of A5 (60.0 mg, 0.040 mmol) in DMSO (3.0 mL) was addedIBX (58.8 mg, 0.21 mmol). The reaction mixture was stirred at 40° C. for16 h. The mixture was purified by prep-HPLC (ACN 40-70%/0.225% FA inwater) to afford (15 mg, 25.1%) as a white solid. LCMS (5-95, AB, 1.5min): RT=0.758 min, m/z=712.5 [M/2+1]+.

In some aspects of the disclosure, PBD dimer disulfide prodrug 4comprising a linker may be conjugated to an antibody to form PBD dimerADC disulfide prodrug 4.

Example 21B: Preparation of PBD Dimer Disulfide Prodrug 3 Comprising aLinker

PBD dimer disulfide prodrug 3 comprising a linker was prepared accordingto the following reaction scheme:

To a solution of triphosgene (486.9 mg, 1.64 mmol) in DCM (10 mL) wasadded a solution of compound A1 (1.40 g, 1.64 mmol) and triethylamine(664 mg, 6.56 mmol) in DCM (10 mL). The mixture was stirred at 8° C. for10 min. The mixture was concentrated to give the crude product (1.48 g,99.6%) as a yellow solid. To a solution of the above crude product (1.41g, 1.56 mmol) in DCM (15 mL) was added a solution of compound A2 (150.0mg, 0.780 mmol) and triethylamine (158 mg, 1.56 mmol) in DCM (6.0 mL).After the mixture was stirred at 8° C. for 1 h, it was concentrated andpurified by flash column chromatography (50% EtOAc in petroleum ether)to give the product compound A3 (300 mg, 30%) as a yellow solid. LCMS(5-95, AB, 1.5 min): RT=1.193 min, m/z=1071.5 [M+1]+.

To a solution of triphosgene (41.6 mg, 0.140 mmol) in DCM (6.0 mL) wasadded a solution of compound A3 (300 mg, 0.280 mmol) and triethylamine(56.7 mg, 0.560 mmol) in DCM (5.0 mL). The mixture was stirred at 8° C.for 15 min. The mixture was concentrated to give the crude product (307mg, 99.9%) as a yellow solid, which was used for next step directly. Toa solution of the crude product (300.0 mg, 0.270 mmol) in DCM (10 mL)was added a solution of MC_VC_PAB (156 mg, 0.270 mmol) and triethylamine(27.7 mg, 0.270 mmol) in DMF (6.0 mL). After the mixture was stirred at8° C. for 12 h, it was concentrated and purified by flash columnchromatography (6% MeOH in DCM) to give the product compound A4 (140 mg,31%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT=1.354 min,m/z=836.4 [M/2+1]+.

A mixture of compound A4 (140.0 mg, 0.080 mmol) in THF (2.0 mL), water(2.0 mL) and acetic acid (3.0 mL) was stirred at 8° C. for 12 h. Themixture was diluted with EtOAc (60 mL) and washed with water (3×50 mL),saturated NaHCO₃ (50 mL), brine (50 mL). The organic layer was dried andconcentrated to give the crude product compound A5 (120 mg, 99%) as ayellow solid. LCMS (5-95, AB, 1.5 min): RT=0.983 min, m/z=722.0[M/2+1]+.

To a solution of compound A5 (140.0 mg, 0.100 mmol) in DMSO (4.0 mL) wasadded IBX (108 mg, 0.390 mmol) at 18° C. The reaction mixture wasstirred at 40° C. for 8 h. The mixture was purified by prep-HPLC (ACN40-70%/0.225% FA in water) to give the product PBD dimer disulfideprodrug 2 comprising a linker (20 mg, 14%) as a white solid. LCMS (5-95,AB, 1.5 min): RT=0.760 min, m/z=719.7 [M/2+1]+.

In some aspects of the disclosure, PBD dimer disulfide prodrug 3comprising a linker may be conjugated to an antibody to form PBD dimerADC disulfide prodrug 2A or PBD dimer ADC disulfide prodrug 2B.

Example 21C: Preparation of PBD Dimer Disulfide Prodrug 2 Comprising aLinker

PBD dimer disulfide prodrug 2 comprising a linker was prepared accordingto the following reaction scheme:

Synthesis of INTA2:

To a solution of compound A8 (3.18 g, 10.24 mmol) in DCM (25.0 mL) wasadded compound A9 (400 mg, 5.12 mmol). The mixture was stirred at 8° C.for 12 h. To the mixture was added MnO₂ (100 mg) and stirred for 10 minand filtered. The filtrate was concentrated and purified by flash columnchromatography (100% DCM) to give compound 2 (620 mg, 2.67 mmol, 52.1%)as a yellow solid.

To a solution of triphosgene (191.6 mg, 0.65 mmol) in DCM (5.0 mL) wasadded a solution of compound A2 (300 mg, 1.29 mmol) and pyridine (306mg, 3.87 mmol) in DCM (5.0 mL). The mixture was stirred at 8° C. for 10min. The resulting mixture was added dropwise to a solution of compoundA1 (1.43 g, 1.68 mmol) and pyridine (306 mg, 3.87 mmol) in DCM (15.0mL). After the mixture was stirred at 8° C. for 30 min, it wasconcentrated and purified by flash column chromatography (50% EtOAc inpetroleum ether) to give the product compound A3 (0.50 g, 0.427 mmol,33.1%) as a yellow oil. LCMS (5-95, AB, 1.5 min): RT=1.077 min,m/z=1111.7 [M+1]+.

To a solution of compound A3 (300 mg, 0.270 mmol) in DCM (10 mL) wasadded compound A4 (205 mg, 2.7 mmol). The mixture was stirred at 8° C.for 10 min. To the mixture was added MnO₂ (100 mg), stirred for 10 minand filtered. The filtrate was concentrated and purified by flash columnchromatography (50% EtOAc in petroleum ether) to give the productcompound A5 (210 mg, 0.204 mmol, 75.4%) as a yellow solid.

To a solution of triphosgene (30.2 mg, 0.100 mmol) in DCM (5.0 mL) wasadded a solution of compound A5 (210 mg, 0.200 mmol) and triethylamine(61.7 mg, 0.610 mmol) in DCM (5.0 mL). The mixture was stirred at 8° C.for 30 min. Then the mixture was added dropwise to a solution ofMC_VC_PAB (139.8 mg, 0.240 mmol) and triethylamine (61.7 mg, 0.610 mmol)in DMF (5.0 mL). The mixture was stirred at 8° C. for 12 h. The mixturewas concentrated and purified by flash column chromatography (8% MeOH inDCM) to give the product compound A6 (140 mg, 0.085 mmol, 41.8%) as ayellow oil. LCMS (5-95, AB, 1.5 min): RT=1.248 min, m/z=816.1[M/2+1]+.

A mixture of compound A6 (140.0 mg, 0.090 mmol) in acetic acid (3.0 mL),THF (2.0 mL) and water (2.0 mL) was stirred at 8° C. for 8 h. Themixture was diluted with EtOAc (60 mL), washed with water (3×50 mL),saturated NaHCO₃ (50 mL), brine (50 mL) and concentrated to give theproduct compound A7 (120 mg, 0.0856 mmol, 99.7%) as a white solid. LCMS(5-95, AB, 1.5 min): RT=0.787 min, m/z=701.6[M/2+1]+.

To a solution of compound A7 (130.0 mg, 0.090 mmol) in DMSO (3.0 mL) wasadded IBX (129.9 mg, 0.460 mmol) at 9° C. The reaction mixture wasstirred at 50° C. for 48 h. The mixture was purified by prep-HPLC (ACN35-65%/0.225% FA in water) to give the product PBD dimer disulfideprodrug 4 comprising a linker (10 mg, 0.0071 mmol, 7.6%) as a whitesolid. LCMS (5-95, AB, 1.5 min): RT=0.728 min, m/z=1397.9[M+1]+.

In some aspects of the disclosure, PBD dimer disulfide prodrug 2comprising a linker may be conjugated to an antibody to form PBD dimerADC disulfide prodrug 4.

Example 21D: Preparation of a PBD Dimer Disulfide Prodrug Comprising aLinker for Conjugation to Form PBD Dimer Diaphorase Prodrug 1A and 1B,and Having the Structure

and the name: 4-(tert-butyldisulfaneyl)benzyl(11aS)-8-((5-(((11aS)-10-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-11-hydroxy-7-methoxy-2-methylene-5-oxo-2,3,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-10(5H)-carboxylate.

A PBD dimer disulfide prodrug comprising a linker was prepared accordingto the following reaction scheme:

Synthesis of INTA2:

To a solution of A7 (250 mg, 1.78 mmol) in 95% EtOH (10 mL) was added A8(2.01 mL, 17.85 mmol). The mixture was cooled to 0° C. and a solution ofiodine (200 mg, 0.79 mmol) in 95% EtOH (10 mL) was added drop-wise untilthe color of the mixture changed from colorless to brown. After it wasstirred for 2 h, saturated NaHCO₃ (2.0 mL) was added at 0° C. until thepH was greater than 7. The solution was concentrated in vacuo. EtOAc (20mL) was added, and the organic layer was washed with 10% NaHCO₃(3×15 mL)and brine. The organic layer was dried over Na₂SO₄, filtered andconcentrated and purified by flash column chromatography (0-32% EtOAc inpetroleum ether) to afford compound A2 (280 mg, 68.8%) as yellow oil. 1HNMR (400 MHz, CDCl3) δ 7.51 (d, J=8.4 Hz, 2H), 7.23 (d, J=8.4 Hz, 2H),4.57 (s, 2H), 2.40 (br, 1H), 1.29 (s, 9H).

To a solution of triphosgene (26 mg, 0.090 mmol) in DCM (1.0 mL) wasadded a solution of compound A2 (50.0 mg, 0.220 mmol) and pyridine (18.0mg, 0.230 mmol) in DCM (4.0 mL) at 0° C. under N₂. After the reactionmixture was stirred at 0° C. under N₂ for 5 min, it was added dropwiseto a solution of pyridine (34.0 mg, 0.430 mmol) and A1 (277 mg, 0.320mmol) at 0° C. under N₂. The reaction mixture was stirred at 20° C.under N₂ for 2 h. Solvent was removed and the residue was purified byprep-TLC (50% EtOAc in petroleum ether, Rf=0.4) to afford compound A3(70 mg, 28.3%) as a yellow foam. LCMS (5-95, AB, 1.5 min): RT=1.328 min,m/z=1108.7 [M+1]+.

To a solution of triphosgene (46.0 mg, 0.160 mmol) in DCM (7.0 mL) wasadded a mixture of compound A3 (400.0 mg, 0.360 mmol) and triethylamine(37.0 mg, 0.370 mmol) in DCM (3.0 mL) at 0° C. under N₂. After thereaction mixture was stirred at 0° C. for 30 min, a solution ofMC-VC-PAB (247.0 mg, 0.430 mmol) and triethylamine (73.0 mg, 0.720 mmol)in DMF (3.0 mL) was added at 20° C. under N₂. The reaction mixture wasstirred at 40° C. under N₂ for 8 h. The mixture was concentrated andpurified by column chromatography (0-6% MeOH in DCM) to afford compoundA4 (190 mg, 30.7%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT=1.308min, m/z=854.2 [M/2+1]+.

To a solution of compound A4 (415 mg, 0.240 mmol) in water (2.0 mL) andTHF (2 mL) was added HOAc (6.47 mL, 113 mmol) at 15° C. After thereaction mixture was stirred at 15° C. for 7 h, it was diluted withEtOAc (30 mL) and washed with water (2×15 mL), saturated aq. NaHCO₃ (15mL) and brine (15 mL). It was dried and concentrated to give the crudecompound A5 (150 mg, 41.7%) as a yellow solid, which used directly forthe next step without further purification. LCMS (5-95, AB, 1.5 min):RT=0.997 min, m/z=739.4 [M/2+1]+.

To a solution of compound A5 (50.0 mg, 0.030 mmol) in DMSO (3.0 mL) wasadded IBX (38.0 mg, 0.140 mmol) at 18° C. The reaction mixture wasstirred at 37° C. for 8 h. The mixture was purified by prep-HPLC (ACN40-70%/0.225% FA in water) to afford PBD dimer disulfide prodrug 1comprising a linker (17.2 mg, 33.1%) as a white solid. LCMS (5-95, AB,1.5 min): RT=0.806 min, m/z=737.1 [M/2+1]+.

In some aspects of the disclosure, the PBD dimer disulfide prodrugcomprising a linker may be conjugated to an antibody to form PBD dimerADC disulfide prodrug 1A or PBD dimer ADC disulfide prodrug 1B.

Example 21E: Preparation of PBD Dimer Disulfide Prodrug 5 Comprising aLinker

PBD dimer disulfide prodrug 5 comprising a linker was prepared accordingto the following reaction scheme:

Synthesis of INTA2:

To a solution of TBDPSCl (8.62 g, 31.36 mmol) in DMF (40 mL) was added asolution of compound A8 (2.27 g, 29.05 mmol) in DMF (30 mL). After thesolution was stirred for 10 min, imidazole (4.27 g, 62.7 mmol) in DMF(8.0 mL) was added and the reaction mixture was stirred at 20° C. for 24h. The mixture was concentrated and taken up in DCM (30 mL), filtered,and washed with H2O (3×30 mL). The organic layer was dried with MgSO₄,filtered, and the solvent was removed. The residue was purified by flashcolumn chromatography (3% EtOAc in petroleum ether) to give compound A9(7.0 g, 76%) as a colorless oil.

To a solution of compound A9 (1.00 g, 3.16 mmol) in DCM (10 mL) wasadded a solution of compound A10 (1.96 mg, 6.32 mmol) in DCM (10 mL)dropwise over 15 min. After the mixture was stirred for another 1 h at26° C., manganese dioxide (1.00 g, 11.5 mmol) was added and stirred for10 min, until the yellow solution became colorless. Manganese dioxidewas filtered off, and the filtrate was concentrated. MeOH (5.0 mL) wasadded and the solid was filtered to remove compound A10. The residue waspurified by column chromatography (0-2.5% EtOAc in petroleum ether) toafford compound A11 (0.90 g, 61%) as a yellow solid.

To a solution of compound A11 (250 mg, 1.89 mmol) in DCM (6.0 mL) wasadded a solution of compound A12 (0.50 g, 1.58 mmol) in DCM (4.0 mL)dropwise over 15 min. After addition, the mixture was stirred foranother 1 h at 26° C. Manganese dioxide (1.0 g, 11.5 mmol) was added.The mixture was stirred for another 10 min, until yellow reactionsolution became colorless. Manganese dioxide was filtered off, thefiltrate was concentrated and MeOH (5.0 mL) was added. Solid wasfiltered off and the residual was purified by column chromatography(0-14% EtOAc in petroleum ether) to afford compound A2 (0.500 g, 71%) asa yellow oil.

To a solution of triphosgene (695 mg, 2.34 mmol) in DCM (5.0 mL) wasadded a solution of compound A1 (2.0 g, 2.34 mmol) and triethylamine(711.0 mg, 7.03 mmol) in DCM (10 mL) at 0° C. The mixture was stirred at0° C. for 30 min. The reaction mixture was concentrated, and a solutionof compound A2 (680 mg, 1.52 mmol) and triethylamine (308 mg, 3.04 mmol)in DCM (4.0 mL) was added. The mixture was stirred at 25° C. for 2 h,concentrated, and purified by column chromatography (0-50% EtOAc inpetroleum ether) to afford A3 (700 mg, 33%) as a yellow solid. LCMS(5-95, AB, 1.5 min): RT=1.401 min, m/z=1326.0 [M+1]+.

To a solution of triphosgene (54.0 mg, 0.180 mmol) in DCM (10 mL) wasadded a mixture of compound A3 (500 mg, 0.450 mmol) and triethylamine(50.0 mg, 0.490 mmol) in DCM (5.0 mL) at 0° C. under N₂. The reactionmixture was stirred at 0° C. for 30 min. To the reaction mixture wasadded a solution of Fmoc-VC_PAB (260.0 mg, 0.450 mmol) and triethylamine(78.0 mg, 0.770 mmol) in DMF (3.0 mL). The mixture was stirred at 40° C.for 8 h, concentrated, and purified by column chromatography (0-8% MeOHin DCM) to give compound A4 (130 mg, 14%) as a yellow solid. LCMS (5-95,AB, 1.5 min): RT=1.474 min, m/z=978.1 [M/2+1]+.

To a solution of compound A4 (130.0 mg, 0.070 mmol) in water (2.0 mL)and THF (2.0 mL) was added HOAc (3.0 mL, 52.5 mmol) at 26° C. andstirred at 26° C. for 7 h. The reaction mixture was diluted with EtOAc(20 mL), washed with water (2×15 mL), saturated NaHCO₃ (10 mL) and brine(10 mL). It was dried and concentrated to give the crude compound A5(130 mg, 83%) as a yellow solid, which used directly for the next stepwithout further purification. LCMS (5-95, AB, 1.5 min): RT=1.065 min,m/z=863.2 [M/2+1]+.

To a solution of compound A5 (130.0 mg, 0.080 mmol) in DMSO (3.0 mL) wasadded and 2-iodoxybenzoic acid (84.4 mg, 0.300 mmol) at 25° C. After themixture was stirred at 40° C. for 10 h, it was purified by prep-HPLC(ACN 85-100%, 0.225% FA in water) to afford product A6 (60 mg, 45%) as awhite solid. LCMS (5-95, AB, 1.5 min): RT=1.178 min, m/z=861.4 [M/2+1]+.

To a solution of compound A6 (60.0 mg, 0.035 mmol) in THF (3.0 mL) wasadded TBAF (39.4 mg, 0.150 mmol) at 26° C. The reaction mixture wasstirred at 26° C. for 2 h. The reaction mixture was diluted with DCM (20mL), washed with water (3×15 mL), dried over Na₂SO₄, and concentrated togive the crude compound A7 (70 mg, crude) as a yellow oil.

To a stirred solution of Compound A7 (70.0 mg, 0.060 mmol) in DMF (2.0mL) was added MC_OSu (51.4 mg, 0.170 mmol) at 26° C. The mixture wasstirred at 26° C. for 2 h. The reaction mixture was purified byprep-HPLC (ACN 35-55%/0.225% FA in water) to afford (5.5 mg, 6.7%) as awhite solid. LCMS (5-95, AB, 1.5 min): RT=0.852 min, m/z=1453.5 [M+1]+.

In some aspects of the disclosure, PBD dimer disulfide prodrug 5comprising a linker may be conjugated to an antibody to form PBD dimerADC disulfide prodrug 5.

Example 22: Preparation of PBD Dimer Boronic Acid Prodrugs Comprising aLinker for Conjugation to an Antibody Example 22A: Preparation of PBDDimer Boronic Acid Prodrug 1 Comprising a Linker

PBD dimer boronic acid prodrug 1 comprising a linker was preparedaccording to the following reaction scheme:

Each asterisk in the above structure, and elsewhere depicted in Example22, represents a chiral center.

To a solution of triphosgene (228 mg, 0.77 mmol) in THF (15 mL) wasadded a solution A2 (450 mg, 1.92 mmol) and pyridine (304 mg, 3.84 mmol)in THF (5.0 mL) at 0° C. under N₂. The mixture was stirred at 0° C. for20 min under N₂ and was added to a solution of A1 (2.04 g, 2.39 mmol)and triethylamine (389 mg, 3.84 mmol) in DCM (20 mL) at 0° C. under N₂.The reaction mixture was stirred at 10° C. for 2 h. The mixture wasconcentrated and purified by prep-TLC (30-60% EtOAc in petroleum ether)to give compound A3 (500 mg, 26%) as a yellow solid. LCMS (5-95, AB, 1.5min): RT=1.268 min, m/z=1113.4 [M+1]+.

To a solution of triphosgene (53 mg, 0.18 mmol) in DCM (15 mL) was addeda mixture of compound A3 (500 mg, 0.39 mmol) and triethylamine (45 mg,0.44 mmol) in DCM (5.0 mL) at 0° C. under N₂. The reaction mixture wasstirred at 0° C. for 30 min. To this reaction mixture was added asolution of MC_VC_PAB (308 mg, 0.54 mmol) and triethylamine (90 mg, 0.89mmol) in DMSO (4.0 mL) at 10° C. under N₂. The reaction mixture wasstirred at 40° C. under N₂ for 6 h. The mixture was diluted with DCM (30mL), washed with water (2×15 mL) and the aqueous layer was extractedwith EtOAc (2×20 mL). The combined organic layer was dried over Na₂SO₄,concentrated and purified by flash column (0-10% MeOH in DCM) to affordcompound 8 (300 mg, 34% yield) as a yellow solid. LCMS (5-95, AB, 1.5min): RT=1.253 min, m/z=857.1 [M/2+1]+.

To a solution of compound A4 (100.0 mg, 0.050 mmol) in water (2.0 mL)and THF (2.0 mL) was added HOAc (3.0 mL) at 10° C. The reaction mixturewas stirred at 10° C. for 6 h. The reaction mixture was diluted withEtOAc (20 mL), washed with water (2×15 mL), saturated aq. NaHCO₃ (15 mL)and brine (15 mL). It was dried over Na₂SO₄ and concentrated to give thecrude compound A5 (71 mg, 99.8% yield) as a yellow solid. LCMS (5-95,AB, 1.5 min): RT=0.829 min, m/z=857.1 [M/2-17]+.

To a solution of compound A5 (36 mg, 0.030 mmol) in DMSO (3.0 mL) wasadded 2-iodoxybenzoic acid (22 mg, 0.080 mmol) at 9° C. The reactionmixture was stirred at 40° C. for 6 h. The mixture was purified byprep-HPLC (ACN 27-47%/0.225% FA in water) to afford PBD dimer boronicacid prodrug 1 comprising a linker (2.0 mg, 5.5%) as a white solid. LCMS(5-95, AB, 1.5 min): RT=0.813 min, HRMS: m/z=1397.5906 [M+1]+.

In some aspects of the disclosure, PBD dimer boronic acid prodrug 1comprising a linker may be conjugated to an antibody to form PBD dimerADC boronic acid prodrug 1A or PBD dimer ADC boronic acid prodrug 1B.

Example 22B: Preparation of PBD Dimer Boronic Acid Control 1 Comprisinga Linker

PBD dimer boronic acid control 1 comprising a linker was preparedaccording to the following reaction scheme:

To compound A1 (500.0 mg, 0.590 mmol) in DMF (2.0 mL) was added Cbz-OSu(161 mg, 0.640 mmol). After the mixture was stirred at 50° C. for 4 h,another batch of Cbz-OSu (161 mg, 0.640 mmol) was added. The mixture wasstirred at 50° C. for another 16 h, and purified by prep-TLC (10% MeMHin DCM, R=0.8), followed by prep-HPLC to afford compound A2 (220 mg,35.2%) as an orange oil. LCMS (5-95, AB, 1.5 min): RT=1.097 min,m/z=987.5 [M+1]+.

To a mixture of triphosgene (26.45 mg, 0.090 mmol) and 4 Å MS (30 mg) inDCM (3.0 mL) was added a solution of compound A2 (220.0 mg, 0.220 mmol)and triethylamine (22.6 mg, 0.220 mmol) in DCM (2.0 mL) at 0° C. Themixture was stirred at 0° C. for 1 h, and concentrated. To the solutionthis isocyanate (225.0 mg, 0.220 mmol) in DCM (6.0 mL) was added asolution of MC_VC_PAB (34.59 mg, 0.060 mmol), Et3N (23 mg, 0.22 mmol)and 4 Å MS (30 mg) in DMF (2.0 mL) at 0° C. After the mixture wasstirred at 20° C. for 16 h, it was quenched with water. DCM (10 mL) wasadded, separated, and DCM phase was concentrated and purified byprep-TLC (15% MeOH in DCM, Rf=0.4) to afford compound A3 (70 mg, 19.8%)as light yellow oil. LCMS (5-95, AB, 1.5 min): RT=1.107 min, m/z=793.9[M/2+1]+.

Compound A3 (70.0 mg, 0.040 mmol) in mixture of THF (3.0 mL) and water(2.0 mL) was added HOAc (1.0 mL, 17.49 mmol), and the mixture stirred at40° C. for 8 h. The mixture was concentrated and purified by prep-TLC(13% MeOH in DCM, Rf=0.5), to afford compound A4 (40 mg, 67.8%) as ayellow oil. LCMS (5-95, AB, 1.5 min): RT=0.751 min, m/z=679.6 [M/2+1]+.

To a solution of compound A4 (30.0 mg, 0.020 mmol) in DMSO (3.0 mL) wasadded 2-iodoxybenzoic acid (30.9 mg, 0.110 mmol) at 18° C. After thereaction mixture was stirred at 40° C. for 16 h, it was purified byprep-TLC (10% MeOH in DCM, Rf=0.4) to afford PBD dimer boronic acidcontrol 1 comprising a linker (13 mg, 43.5%) as a white solid. LCMS(5-95, AB, 1.5 min): RT=0.726 min, m/z=677.5 [M/2+1]+.

In some aspects of the disclosure, PBD dimer boronic acid control 1comprising a linker may be conjugated to an antibody to form PBD dimerADC boronic acid control 1A or PBD dimer ADC boronic acid control 1B.

Example 23: Preparation of PBD Monomer and Dimer Diaphorase ProdrugsExample 23A: Preparation of PBD Monomer Diaphorase Prodrug 2

PBD monomer diaphorase prodrug 2 was prepared according to the followingreaction scheme:

Each asterisk in the above structure, and elsewhere depicted in Example23, represents a chiral center.

To a solution of triphosgene (58.39 mg, 0.200 mmol) in DCM (2.0 mL) at0° C. was added solution of compound A1 in DCM (5.0 mL) under N₂ slowly,and the mixture stirred at 0° C. for 1 h. Compound A2 (100.0 mg, 0.450mmol) and triethylamine (91.5 mg, 0.900 mmol) in DCM (2.0 mL) was addedto above solution at 0° C. dropwise over 10 min. The mixture was stirredat 0° C. for 1 h. The mixture was quenched with water (5.0 mL),separated and concentrated. It was purified by prep-TLC (5% MeOH in DCM,Rf=0.7) to afford compound A3 (63 mg, 19%) as orange solid. LCMS (5-95,AB, 1.5 min): RT=1.007 min, m/z=654.3 [M+1]+.

To a solution of Compound A3 (63.0 mg, 0.100 mmol) THF (2.0 mL) andwater (1.0 mL) was added acetic acid (2.0 mL, 2.1 mmol). The mixture wasstirred at 15° C. for 16 h. The mixture was concentrated and purified byprep-TLC (5% MeOH in DCM, Rf=0.3) to afford A4 (40 mg, 77%) as orangesolid. LCMS (5-95, AB, 1.5 min): RT=0.761 min, m/z=540.1 [M+1]+.

Compound A4 (26.0 mg, 0.050 mmol) in DCM (3.0 mL) was added DMP (40.0mg, 0.090 mmol), the mixture was allowed to stir at 10° C. for 16 h. Themixture was quenched with mixture of saturated Na₂SO₃ and NaHCO₃solution (3.0 mL/3.0 mL). Organic phase was separated, concentrated, andpurified by prep-HPLC (Diamonsil 150*20 mm*5 um, 0.225% FA-ACN, ACN23-53%) to afford PBD monomer diaphorase prodrug 2 (20 mg, 74%) asorange solid. LCMS (5-95, AB, 1.5 min): RT=0.747 min, m/z=538.1 [M+1]+.

1H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 6.64 (s, 1H), 6.52 (s, 1H), 5.69(s, 1H), 5.59-5.57 (d, J=9.6 Hz, 1H), 5.30-5.27 (d, J=13.6 Hz, 1H),5.16-5.15 (m, 2H), 4.94-4.90 (d, J=13.6 Hz, 1H), 4.32-4.27 (d, J=16.4Hz, 1H), 4.17-4.13 (d, J=16.4, 1H), 3.93 (s, 3H), 3.83 (s, 3H),3.78-3.76 (m, 6H), 3.64-3.59 (m, 1H), 2.47 (m, 1H), 2.96-2.89 (m, 1H),2.73-2.69 (m, 1H).

Example 23B: Preparation of PBD Dimer Diaphorase Prodrug 1

PBD dimer diaphorase prodrug 1 was prepared according to the followingreaction scheme:

To a solution of triphosgene (56.03 mg, 0.190 mmol) in DCM (2.0 mL) wasadded a solution of compound A1 (400 mg, 0.420 mmol) and triethylaminein DCM (5.0 mL) dropwise at 0° C. After the mixture was stirred for 1 hat 19° C., a solution of compound A2 (92.8 mg, 0.420 mmol) andtriethylamine (42.5 mg, 0.420 mmol) in DMSO (0.50 mL)/DCM (2.50 mL) wasadded. The mixture was stirred at 19° C. for 2.0 h. The mixture wasdiluted with DCM (20.0 mL), washed with water (2×10.0 mL), andseparated. The aqueous layer was extracted with EtOAc (2×20 mL), and thecombined organic layer was dried over Na₂SO₄, concentrated and purifiedby prep-TLC (20% MeOH in DCM, Rf=0.7) to afford compound A3 (150 mg,30%). LCMS (5-95, AB, 1.5 min): RT=1.118 min, m/z=1201.7 [M+1]+.

To a solution of compound A3 (130.0 mg, 0.110 mmol) in THF (2.0 mL) wasadded acetic acid (3.0 mL) and water (1.0 mL). The mixture was stirredat 18° C. for 18 h. Saturated NaHCO₃ was added to adjust pH=8, and themixture was extracted with DCM (2×50 mL). The organic layer wascombined, washed with brine (30 mL) and water (30 mL), and dried overNa₂SO₄. It was concentrated and purified by prep-TLC (6.7% MeOH in DCM)to afford compound A4 (72 mg, 68%) as yellow solid. LCMS (5-95, AB, 1.5min): RT=0.766 min, m/z=972.3 [M+1]+.

To a solution of compound 4 (50.0 mg, 0.050 mmol) in DCM (10 mL) wasadded DMP (76.36 mg, 0.180 mmol), and the mixture was stirred at 18° C.for 18 h. The mixture was filtered, and the filtrate was washed withsaturated Na₂CO₃ (20.0 mL). The aqueous layer was extracted with DCM(2×20.0 mL), and the organic layer was combined, dried over Na₂SO₄ andconcentrated. It was purified by prep-TLC (6.7% MeOH in DCM, Rf=0.5) togive compound A5 (40 mg, 74%) as yellow solid. LCMS (5-95, AB, 1.5 min):RT=0.731 min, m/z=990.2 [M+23]+.

TFA (1.0 mL) was added dropwise to compound A5 (40.0 mg, 0.040 mmol) at0° C. The mixture was stirred at 0° C. for 30 min. Saturated NaHCO₃ wasadded to the mixture dropwise at 0° C. to adjust pH=7. It was extractedwith DCM (3×20.0 mL), and the combined organic layer was washed withbrine (20.0 mL), dried over Na₂SO₄ and concentrated. The residue waspurified by prep-TLC (6.7% MeOH in DCM, Rf=0.6) to afford PBD dimerdiaphorase prodrug 1 (4.9 mg, 14%) as yellow solid. LCMS (5-95, AB, 1.5min): RT=0.807 min, m/z=850.2 [M+23]+.

Example 23C: Preparation of PBD Monomer Diaphorase Prodrug 3

PBD monomer diaphorase prodrug 3 was prepared according to the followingreaction scheme:

To a solution of compound A1 (280 mg, 1.27 mmol) in DMF (10 mL) wasadded DIEA (490 mg, 3.79 mmol) and bis (4-nitrophenyl) carbonate (770mg, 2.53 mmol) at 16° C. The reaction mixture was stirred at 16° C.under N₂ for 2 h. The reaction solution was concentrated and washed withMTBE to afford compound A2 (500 mg, 78%) as an orange solid. LCMS (5-95,AB, 1.5 min): RT=0.726 min, m/z=491.0 [M+23]+.

To a solution of compound A2 (500 mg, 0.99 mmol), compound A3 (240 mg,1.95 mmol) and HOBt (13 mg, 0.10 mmol) in DMF (8.0 mL) was added DIEA(340 mg, 2.63 mmol) at 16° C. The reaction mixture was stirred at 50° C.under N₂ for 2 h. The reaction was concentrated and the residue waswashed by MeCN (3×8 mL) to give compound A4 (350 mg, 95%) as an orangesolid. LCMS (5-95, AB, 1.5 min): RT=0.745 min, m/z=393.1 [M+23]+.

To a solution of triphosgene (58 mg, 0.20 mmol) in DCM (8.0 mL) wasadded a solution of compound A5 (200 mg, 0.49 mmol) and triethylamine(60.0 mg, 0.59 mmol) in DCM (1.5 mL) at 0° C. under N₂. The reactionmixture was stirred at 12° C. under N₂ for 30 min and a solution ofcompound A4 (91 mg, 0.25 mmol), TEA (75 mg, 0.74 mmol) and DMAP (6 mg,0.05 mmol) in DCM (1.5 mL) and DMSO (1.0 mL) at 0° C. was added underN₂. After the reaction mixture was stirred at 12° C. under N₂ for 6 h,it was diluted with DCM (30 mL), washed with water (2×15 mL). Theaqueous layer was extracted with EtOAc (2×20 mL), and the combinedorganic layer was dried over Na₂SO₄, concentrated, and purified byprep-HPLC (ACN 66-86%/0.225% FA in water) to afford compound A6 (60 mg,12%) as an orange solid. LCMS (5-95, AB, 1.5 min): RT=1.051 min,m/z=803.2 [M+1]+.

To a solution of compound A6 (40 mg, 0.05 mmol) in water (1.0 mL) andTHF (1.0 mL) was added HOAc (1.5 mL, 26 mmol) at 10° C. The reactionmixture was stirred at 10° C. for 6 h. The reaction mixture was dilutedwith EtOAc (20 mL) and washed with water (2×15 mL), saturated NaHCO₃ (15mL) and brine (15 mL). It was dried over Na₂SO₄, concentrated, andpurified by prep-TLC (6.25% MeOH in DCM) to afford compound A7 (35 mg,97%) as an orange solid. LCMS (5-95, AB, 1.5 min): RT=0.819 min,m/z=689.1 [M+1]+.

To a solution of compound A7 (35 mg, 0.050 mmol) in DCM (4.0 mL) wasadded DMP (61 mg, 0.14 mmol) at 0° C. The reaction mixture was stirredat 10° C. for 10 h. The reaction was quenched with saturatedNaHCO₃/Na₂SO₃ (4.0 mL/4.0 mL) and extracted with DCM (3×10 mL). Thecombined organic layer was washed with NaHCO₃/Na₂SO₃ (4.0 mL/4.0 mL),brine (7.0 mL). It was dried over Na₂SO₄, concentrated, and purified byprep-HPLC (ACN 30-60%/0.225% FA in water) to afford PBD monomerdiaphorase prodrug 3 (15 mg, 45%) as an orange solid. LCMS (5-95, AB,1.5 min): RT=0.806 min, m/z=687.2 [M+1]+; 1H NMR (400 MHz, CDCl3) δ7.30-7.25 (m, 2H), 7.18-7.15 (M, 4H), 6.77 (s, 1H), 6.69 (S, 1H), 6.50(s, 1H), 5.67 (s, 1H), 5.56 (d, J=9.2 Hz, 1H), 5.27 (d, J=12.4 Hz, 1H),5.17-5.12 (m, 4H), 4.81 (d, J=12.4 Hz, 1H), 4.27 (d, J=16.0 Hz, 1H),4.12 (d, J=16.0 Hz, 1H), 3.33 (s, 3H), 3.89 (s, 3H), 3.81 (s, 3H),3.68-3.59 (m, 4H), 2.93-2.87 (m, 1H), 2.69 (d, J=15.6 Hz, 1H).

Example 23D: Preparation of PBD Dimer Diaphorase Prodrug 2

PBD dimer diaphorase prodrug 2 was prepared according to the followingreaction scheme:

To a solution of triphosgene (62.0 mg, 0.210 mmol) in DCM (12 mL) wasadded a solution of compound A1 (500.0 mg, 0.520 mmol) and triethylamine(63.0 mg, 0.620 mmol) in DCM (2.0 mL) at 0° C. under N₂. The reactionmixture was stirred at 12° C. under N₂ for 30 min, and a solution ofcompound A2 (97.0 mg, 0.260 mmol), DMAP (6.0 mg, 0.050 mmol) andtriethylamine (79.0 mg, 0.780 mmol) in DCM (1.5 mL) and DMSO (0.60 mL)was added at 0° C. under N₂. The reaction mixture was stirred at 12° C.under N₂ for 6 h. It was diluted with DCM (30 mL), washed with water(2×15 mL). The aqueous layer was extracted with EtOAc (2×20 mL), and theorganic layer was dried over Na₂SO₄. It was purified by silicachromatography (3% MeOH in DCM) to afford compound A3 (200 mg, 49%) asan orange solid. LCMS (5-95, AB, 1.5 min): RT=1.294 min, m/z=1349.6[M+1]+.

To a solution of compound A3 (206.9 mg, 0.130 mmol) in water (2.0 mL)and THF (2.0 mL) was added HOAc (3.0 mL, 52.46 mmol) at 9° C. Thereaction mixture was stirred at 9° C. for 10 h. It was diluted with DCM(20 mL), and the mixture was washed with NaHCO₃ (2×15 mL), water (15mL). It was dried over Na₂SO₄, concentrated, and purified by prep-TLC(6.25% MeOH in DCM) to afford compound A4 (100 mg, 66%) as a yellowsolid. LCMS (5-95, AB, 1.5 min): RT=0.932 min, m/z=1121.6 [M+1]+.

To a solution of compound A4 (100.0 mg, 0.090 mmol) in DCM (10 mL) wasadded DMP (113.0 mg, 0.270 mmol) at 0° C. The reaction mixture wasstirred at 9° C. for 10 h. The reaction was quenched with a saturatesolution of NaHCO₃/Na₂SO₃ (5.0 mL/5.0 mL) and extracted with DCM (3×10mL). The combined organic layer was washed with NaHCO₃/Na₂SO₃ (5 mL/5mL), brine (10 mL), dried and concentrated. The residue was purified byprep-TLC (6.25% MeOH in DCM) to give compound A5 (45 mg, 46%) as anorange solid. LCMS (5-95, AB, 1.5 min): RT=0.884 min, m/z=1140.0[M+23]+.

Cold TFA (95% in water, 2.0 mL) was added to compound A5 (35.0 mg, 0.030mmol) at 0° C. The reaction mixture was stirred at 0° C. for 15 min. Thereaction mixture was added dropwise to a saturate aq. NaHCO₃ (4.0 mL) at0° C. and extracted with DCM (4×8.0 mL). The combined organic layer waswashed with brine (15 mL), dried over Na₂SO₄, and concentrated. It waspurified by prep-HPLC (ACN 36-66/0.225% FA in water) to afford PBD dimerdiaphorase prodrug 2 (6.1 mg, 19%) as an orange solid. LCMS (5-95, AB,1.5 min): RT=0.831 min, m/z=999.3 [M+1]+.

Example 23E: Preparation of PBD Dimer Diaphorase Prodrug 3

PBD dimer diaphorase prodrug 3 was prepared according to the followingreaction scheme:

To a solution of triphosgene (28.0 mg, 0.090 mmol) in DCM (5.0 mL) wasadded a solution of compound A1 (200 mg, 0.210 mmol) and triethylamine(42.0 mg, 0.420 mmol) in DCM (3.0 mL) at 0° C. It was stirred at 26° C.for 20 min under N₂. The mixture was concentrated and redissolved in DCM(3.0 mL), and added to a stirred solution of triethylamine (38.0 mg,0.380 mmol) and compound A2 (51.0 mg, 0.230 mmol) in DCM (3.0 mL) at 0°C. It was stirred at 26° C. for 2 h under N₂, and purified by columnchromatography (0-40% EtOAc in petroleum ether) to afford compound A3(240 mg, 90%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT=1.159 min,m/z=1200.5 [M+1]+.

A solution of compound A3 (240.0 mg, 0.200 mmol) in THF (4.0 mL) andwater (4.0 mL) was added HOAc (6.0 mL, 153 mmol) at 25° C. The mixturewas stirred for 10 h at 25° C., and EtOAc (100 mL) was added. It waswashed with water (50 mL), saturated NaHCO₃ (50 mL), then brine (50 mL).The organic layer was dried and concentrated to give the crude productcompound A4 (194 mg, 99.8%) as a yellow solid. LCMS (5-95, AB, 1.5 min):RT=0.767 min, m/z=972.4 [M+1]+.

To a solution of compound A4 (194.0 mg, 0.200 mmol) in DCM (15 mL) wasadded DMP (211.6 mg, 0.500 mmol). The mixture stirred at 26° C. for 1 h,and was quenched with saturated Na₂SO₃/NaHCO₃ (10 mL/10 mL). It wasdiluted with DCM (2×30 mL), and separated. DCM phase was washed withwater (20 mL), dried over Na₂SO₄, concentrated and purified by prep-HPLC(ACN 43-63/0.225% FA in water) to afford compound A5 (70 mg, 36% yield)as a yellow solid. LCMS (5-95, AB, 1.5 min): RT=0.733 min, m/z=968.6[M+1]+.

TFA (0.20 mL, 2.68 mmol) was added to compound A5 (40.0 mg, 0.040 mmol)at 0° C. The reaction mixture was stirred at 0° C. for 30 min, and wasquenched with cool saturated NaHCO₃ (2.0 mL). DMSO (2.0 mL) was added,and the mixture was purified by prep-HPLC (ACN 36-66%/10 mM NH4HCO3 inwater) to afford PBD dimer diaphorase prodrug 3 (9.9 mg, 28%) as ayellow solid. LCMS (5-95, AB, 1.5 min): RT=0.695 min, m/z=850.3 [M+1]+.

Example 24: Synthesis of Quinones Example 24A: Synthesis of Quinone 1

Quinone 1 was prepared according to the following reaction scheme:

To a suspension of NaH (8.9 g, 0.223 mol) in DMF (300 mL) was cooled inan ice-bath. To this, a solution of the starting amine (30 g, 0.171 mol)in DMF (150 mL) was added dropwise. The reaction mixture was stirred atrt for 60 minutes. Then iodomethane (31.5 g, 0.223 mol) was added. Thereaction mixture was stirred at rt for 1 hour. Then the mixture waspoured onto 10% aqueous solution of NaHCO₃, extracted with EA. Thecombined organic phases were washed with 10% aqueous solution of NaHCO₃,brine and dried. The solution was concentrated to get crude product,which was triturated from EA/Hex to afford product as a light-yellowsolid (29.5 g, 91.2%).

To a solution of the starting aldehyde (29.5 g, 156 mmol, 1 eq.)5-methoxy-1-methyl-1H-indole-3-carbaldehyde in acetic acid (300 mL),cooled to 10° C. To this, a mixture of nitric acid (4.6 mL) in aceticacid (20 mL) was added. The reaction mixture was then stirred at rt for16 h. A yellow suspension was obtained which was poured on to anice-water mixture and the crystals obtained were filtered off and dried.Crude product was triturated from EA/Hex to afford product as a yellowsolid (30.0 g, 82.1%).

To suspension of the starting material (10 g, 43 mmol) in ethanol (600mL) was added tin powder (44.23 g, 0.37 mol). Followed by the additionof 3 N HCl (200 mL). The reaction was stirred at rt for 2 hours. Thesolution was diluted with saturated aqueous NaHCO₃. The mixture wasfiltered and washed with EA. The organic phase was separated, andaqueous was extracted with EA. The combined organic phase was washedwith brine, dried and concentrated to get crude product, which wastriturated from EA/Hex=1/20 to afford product as a gray solid (6.0 g,68.3%).

The starting aldehyde (5.0 g, 24.5 mmol, 1 eq.) was dissolved in 100 mLTHF. To a suspension of LiAlH₄ (1.86 g, 49 mmol, 2 eq.) in 200 mL THFand cooled to 0° C. The solution of aldehyde was added to the LiAlH₄solution dropwise. The reaction was allowed to reach rt and stirred for30 min at rt. It was quenched with water and then filtered throughcelite, dried with MgSO₄ and evaporated. The residue was used directlyin the next reaction. The residue was solved in 300 mL of acetone. To300 mL of a 0.3 M solution of NaH₂PO₄, 19.7 g (73.5 mmol, 3 eq.) ofFremy's salt was added. This mixture was added to the residue in acetoneand stirred at rt for 0.5 h. The excess acetone was removed in vacuo.The resulting residue was extracted with dichloromethane and washed withwater. The organic layer was dried (MgSO₄) and evaporated. Crude productwas triturated from EA/Hex to get product as an orange solid (2.51 g,46.1%, two steps).

1H NMR (400 MHz, CDCl3) δ 6.70 (s, 1H), 5.69 (s, 1H), 4.64 (d, J=6.9 Hz,2H), 3.93 (s, 3H), 3.84 (s, 3H), 3.78 (t, J=7.0 Hz, 1H).

Example 24B: Synthesis of Quinone 2

Quinone 2 was prepared according to the following reaction scheme:

4.6 g Silica gel was added to ethyl 3-oxobutanoate (50 g, 0.38 mmol). Tothis, methylamine solution (aqueous; 40%, 35.7 g, 0.46 mol) was addedand the mixture stirred overnight. The reaction mixture was extractedwith DCM, dried with MgSO₄, filtered and evaporated to get product ascolourless oil (50 g, 92%).

50 g (0.35 mol 1 eq.) of the imine and 37.7 g (0.35 mol 1 eq.)1,4-benzoquinone is dissolved in 400 mL nitromethane. The mixture isleft for 24 hrs (no stirring). Crystals of product precipitate. Theywere filtered, washed with nitromethane and recrystallized from EtOAc.Yellow solid, yield: 25 g, 30.6%.

To a solution of the alcohol (25 g, 0.11 mol) and KOH (25.5 g, 0.46 mol)in DMSO (200 mL) was stirred at rt for 30 minutes. Then iodomethane (62g, 0.44 mol) was added. The mixture was diluted with EA (700 mL), washedwith 1 N HCl, brine and dried. The solution was concentrated to getcrude product, which was purified by silica gel column to afford productas a gray solid (20 g, 75.5%).

To a solution of 20 g (81 mmol, 1 eq.) ethyl5-methoxy-1,2-dimethyl-1H-indole-3-carboxylate in acetic acid (200 mL),cooled to 0° C. was added a mixture of nitric acid (4.6 mL) and aceticacid (20 mL). The mixture was then stirred at rt for 2 h. A yellowsuspension was obtained which was poured on to an ice-water mixture andthe crystals obtained were filtered off and dried. Crude product waspurified by flash chromatography to afford product as a yellow solid(14.0 g, 59.3%).

To suspension of the starting ester (8.0 g, 27 mmol) in ethanol (600 mL)was added tin powder (14.6 g, 0.123 mol). Followed by the addition of 3N HCl (200 mL). The reaction was stirred at rt for 2 hours. The solventswere removed, and the residue was diluted with water, neutralized withsaturated NaHCO₃. The mixture was filtered and washed with EA. Theorganic phase was separated, and aqueous was extracted with EA. Thecombined organic phase was washed with brine, dried and concentrated toget crude product, which was triturated from EA/Hex=1/20 to affordproduct as a gray solid (5.0 g, 70.6%).

5.0 g (19.06 mmol, 1 eq.) of the starting material was dissolved in 50mL THF. 2.9 g (76.25 mmol, 4 eq.) LiAlH₄ was solved in 250 mL THF andcooled to 0° C. The solution of starting material was added to theLiAlH4 solution dropwise. The reaction was allowed to reach RT andstirred for 30 min at rt. It was quenched with water, NaOH and silicagel. It was filtered through celite, dried with MgSO₄ and evaporated.The residue was used directly in the next reaction. The residue wassolved in 330 mL of acetone. To 330 mL of a 0.3 M solution of NaH₂PO₄15.32 g (57.18 mmol, 3 eq.) of Fremy's salt was added. This mixture wasadded to the hydroxymethyl indole in acetone and stirred at rt for 1 h.The excess acetone was removed in vacuo. The resulting residue wasextracted with dichloromethane and washed with water. The organic layerwas dried (MgSO₄) and evaporated. Crude product was purified by flashchromatography to get product as a red solid (2.51 g, 56%).

1H NMR (400 MHz, CDCl3) δ 5.63 (s, 1H), 4.61 (s, 2H), 3.88 (s, 3H), 3.82(s, 3H), 2.23 (s, 3H).

Example 24C: Synthesis of Quinone 3

Quinone 3 was prepared according to the following reaction scheme:

4-methoxyaniline (32 g, 259.2 mmol) was solved in HCl (37%,64 mL) andwater (112 mL). A solution of NaNO₂ (19.5 g, 283.2 mmol) in water (32mL) was added drop wise at −5° C. After addition, the mixture wasstirred at 0° C. for 15 min and brought to pH 3-4 by addition ofCH₃COONa (16.8 g, 204.8 mmol). Ethyl-2-ethylacetoacetate (44.8 g, 283.2mmol) was solved in ethanol (200 mL) at 0° C. To this solution asolution of KOH (15.6 g, 283.2 mmol) in water (24 mL) was added. Theresulting solution was treated with 320 g of ice. The diazonium salt of4-methoxyaniline was immediately added. The mixture was then adjusted topH 5-6 and stirred at 0° C. for 4 h. The solution was stored at 4° C.overnight and extracted with EA (4×200 mL). The combined extracts werewashed with brine and dried by NaSO₄. Most of solvent was evaporated andthe residue was directly used in next reaction (180 mL).

(Z)-ethyl 2-(2-(4-methoxyphenyl)hydrazono)butanoate (180 mL) was addeddropwise to a solution of 3M HCl/EtOH (180 mL) at 80° C. After addition,the mixture was held at 80° C. for 3 h. The solvent was evaporated, andthe residue was treated with water (60 mL) and DCM (300 mL). the aqueouslayers were then extracted with DCM (3×100 mL). The combined organiclayers were washed with brine (150 mL), dried over Na₂SO₄ and evaporatedto dryness to give crude compound, which was triturated from Hex toafford product as a yellow solid (32.0 g, 53.2%).

Ethyl 5-methoxy-3-methyl-1H-indole-2-carboxylate (20.0 g, 85.7 mmol) wasdissolved in DCM (200 mL) and the mixture was cooled to −20° C., HNO₃(70%, 9 mL) was added and the mixture was stirred for 20 min. It wasneutralized with NaHO₃ and extracted with DCM (3×100 mL), dried withNaSO₄ and concentrated to dryness. Crude product was purified by flashchromatography to afford product as a yellow solid (15.5 g, 59.3%).

MeI (30 mL) was added to ethyl5-methoxy-3-methyl-4-nitro-1H-indole-2-carboxylate (14.5 g, 52.1 mmol)in acetone (500 mL) containing KOH (10 g. 178 mmol). After addition, themixture was stirred at room temperature for 1 h. The solvent wasdecanted from the excess KOH, and the mixture was neutralized with HCl.Then the mixture was extracted with DCM (3×100 mL). The combined organiclayers were washed with brine (150 mL), dried over Na₂SO₄ and evaporatedto dryness to give crude compound, which was triturated from Hex and DCMto afford product as a yellow solid (14.0 g, 53.2%).

Tin powder (9.14 g, 77 mol) was added to a suspension of ethyl5-methoxy-1,3-dimethyl-4-nitro-1H-indole-2-carboxylate (5.0 g, 17.1mmol) in ethanol (500 mL). 3 N HCl (130 mL) was then added. The reactionwas stirred at rt for 2 hours. The solvent was removed, and the residuewas diluted with water and neutralized with saturated NaHCO₃. Themixture was filtered and washed with DCM. The organic phase wasseparated, and the aqueous phase was extracted with DCM. The combinedorganic phase was washed with brine, dried and concentrated to get crudeproduct, which was triturated from EA/Hex=1/20 to afford product as agray solid (4.0 g, 80%).

Ethyl 4-amino-5-methoxy-1,3-dimethyl-1H-indole-2-carboxylate (1.5 g,5.72 mmol) was dissolved in 90 mL THF. 0.88 g (22.68 mmol, 4 eq.) LiAlH4was dissolved in 180 mL THF and cooled to 0° C. The solution of ethyl4-amino-5-methoxy-1,3-dimethyl-1H-indole-2-carboxylate was added to theLiAlH₄ solution dropwise. The reaction was allowed to reach rt andstirred for 30 min at rt. It was then quenched with water, NaOH andsilica gel. It was then filtered through celite, dried with MgSO₄ andevaporated. The residue was dissolved in 170 mL of acetone and was useddirectly in the next reaction. To 180 mL of a 0.3 M solution of NaH₂PO₄,Fremy's salt (4.6 g, 17.16 mmol, 3 eq.) was added. This mixture wasadded to the hydroxymethyl indole in acetone and stirred at rt for 1 h.The excess acetone was removed in vacuo. The resulting residue wasextracted with dichloromethane and washed with water. The organic layerwas dried (MgSO₄) and evaporated. The crude product was purified byflash chromatography to get product as an orange solid (1.0 g, 56%).

1H NMR (400 MHz, CDCl3) δ 5.63 (s, 1H), 4.65 (s, 2H), 4.02 (s, 3H), 3.81(s, 3H), 2.34 (s, 3H).

Example 24D: Synthesis of Quinone 4

Quinone 4 was prepared according to the following reaction scheme:

Into a 250-mL round-bottom flask was placed a solution of5-methoxy-1H-indole-2-carboxylic acid (10 g, 52.31 mmol, 1.00 equiv) inmethanol (100 mL), followed by the addition of thionyl chloride (12.5 g,105.07 mmol, 2.00 equiv) dropwise with stirring. The resulting solutionwas heated to reflux for 4 h, cooled to room temperature andconcentrated under vacuum to afford 11 g (crude) of methyl5-methoxy-1H-indole-2-carboxylate as a gray solid.

Into a 5-L 4-necked round-bottom flask, purged and maintained with aninert atmosphere of nitrogen, was placed N,N-dimethylformamide (2 L),followed by the addition of sodium hydride (37.6 g, 1.10 mol, 1.50equiv, 70%) in several batches with stirring. To this was added methyl5-methoxy-1H-indole-2-carboxylate (150 g, 730.96 mmol, 1.00 equiv)dropwise with stirring at less than 10° C. The mixture was stirred for0.5 h. To the mixture was added MeI (125 g, 0.88 mol, 1.20 equiv)dropwise with stirring. The resulting solution was stirred overnight atroom temperature and diluted with 5 L of water. The solids werecollected by filtration, washed with 3×1 L of water and dried to afford163 g (crude) of methyl 5-methoxy-1-methyl-1H-indole-2-carboxylate as ayellow solid.

Into a 5000-mL 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of LiAlH₄ (111 g,2.92 mol, 4.00 equiv) in tetrahydrofuran (1500 mL), followed by theaddition of a solution of methyl5-methoxy-1-methyl-1H-indole-2-carboxylate (160 g, 729.81 mmol, 1.00equiv) in tetrahydrofuran (1000 mL) dropwise with stirring at 0° C. over30 min. The mixture was stirred at 0° C. for 1 h and at room temperaturefor 3 h. The mixture was then quenched by the addition of 111 g ofwater, 333 mL of aqueous NaOH (15%) and 111 g of water at 0° C. Thesolids were filtered out. The filtrate was dried over anhydrous sodiumsulfate and concentrated under vacuum to afford 100 g (72%) of(5-methoxy-1-methyl-1H-indol-2-yl)methanol as a yellow solid.

Into a 3000-mL 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of(5-methoxy-1-methyl-1H-indol-2-yl)methanol (100 g, 522.94 mmol, 1.00equiv) in dichloromethane (2000 mL), followed by the addition oftriethylamine (61.6 g, 608.76 mmol, 1.50 equiv) dropwise with stirringat rt. The mixture was stirred for 30 min. To this was added acetylchloride (79 g, 1.01 mol, 1.50 equiv) dropwise with stirring at roomtemperature. The resulting solution was stirred at room temperature for3 h and concentrated under vacuum. The residue was purified on a silicagel column eluting with ethyl acetate: petroleum ether (1:10-1:5) toafford 75 g (61%) of (5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate asa yellow solid.

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed N,N-dimethylformamide (20 g,273.64 mmol, 6.00 equiv), followed by the addition of POCl₃ (9.85 g,0.0642 mol, 1.50 equiv) dropwise with stirring at 0° C. The mixture wasstirred at room temperature for 30 min. To this was added(5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate (10 g, 42.87 mmol, 1.00equiv) in portions with stirring at less than 0° C. The resultingsolution was stirred at room temperature for 2 h and quenched by theaddition of 100 mL of water/ice. The pH value of the solution wasadjusted to 7-8 with aqueous sodium hydroxide (2 N). The resultingsolution was extracted with 3×200 mL of ethyl acetate. The organiclayers were combined, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluting with ethyl acetate: petroleum ether (1:3) to afford 9 g(80%) of (3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate as ayellow solid.

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed(3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate (9 g, 3.60mmol, 1.00 equiv), AcOH (100 mL), followed by the addition of a solutionof HNO3 (20 mL) in AcOH (50 mL) dropwise with stirring at less than 5°C. The resulting solution was stirred at room temperature for 30 min,diluted with 1000 mL of water and stirred for 30 min. The solids werecollected by filtration, washed with 3×100 mL of water and dried toafford 8.6 g (crude) of(3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl)methyl acetate as alight red solid.

Into a 1000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed(3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl)methyl acetate (8 g,26.12 mmol, 1.00 equiv), ethanol (400 mL), followed by the addition ofSn (34.1 g, 11.00 equiv) in portions with stirring at 0° C. To this wasadded hydrogen chloride (4 N) (400 mL) dropwise with stirring. Theresulting solution was stirred at 0° C. for 2 h, concentrated undervacuum and diluted with 500 mL of water. The pH value of the solutionwas adjusted to 7-8 with saturated aqueous sodium bicarbonate. Thesolids were filtered out and washed with 3×50 mL of EA. The filtrate wasextracted with 4×200 mL of ethyl acetate. The organic layers werecombined, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluting withethyl acetate: petroleum ether (1:2) to afford 6.5 g (90%) of(4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate as ayellow solid.

Into a 2000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed(4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate (6 g,21.72 mmol, 1.00 equiv), acetone (600 mL), followed by the addition of asolution of (KO₃S)₂NO (17.48 g, 65.2 mol, 3.00 equiv) in NaH₂PO₄ (0.4 M)(1200 mL) dropwise with stirring at less than 10° C. The resultingsolution was stirred at room temperature for 2 h and concentrated undervacuum. The residue was extracted with 3×300 mL of dichloromethane. Theorganic layers were combined, washed with 3×300 mL of water, dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluting with ethyl acetate: petroleumether (1:2) to afford 3.5 g (55%) of(3-formyl-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indol-2-yl)methylacetate as a yellow solid.

Into a 500-mL 3-necked round-bottom flask was placed(3-formyl-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indol-2-yl)methylacetate (3.5 g, 12.02 mmol, 1.00 equiv), CH₃CN (200 mL), NaH₂PO₄ (0.6 g,4 mmol, 0.30 equiv), H₂O₂(2 g, 59 mmol, 5.00 equiv), NaClO₂ (2.5 g, 28mmol, 2.41 equiv), and H₂O (50 mL). The resulting solution was stirredat room temperature for 2 h and quenched by the addition of 500 mL ofwater. The pH value of the solution was adjusted to 2 with HCl (2mol/L). The resulting solution was extracted with 4×300 mL of ethylacetate. The organic layers were combined, washed with 2×200 mL ofbrine, dried over anhydrous sodium sulfate and concentrated under vacuumto afford 3.5 g (95%) of2-[(acetyloxy)methyl]-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indole-3-carboxylicacid as a red solid.

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed2-[(acetyloxy)methyl]-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indole-3-carboxylicacid (3.8 g, 12.37 mmol, 1.00 equiv), methanol (100 mL), and hydrogenchloride (20 mL). The resulting solution was stirred at 60° C. for 4 h,concentrated under vacuum, quenched by the addition of 500 mL of waterand extracted with 4×200 mL of ethyl acetate. The organic layers werecombined, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluting withethyl acetate: petroleum ether (1:3). The crude product was purified byPrep-HPLC to afford 0.3 g (9%) of methyl2-(hydroxymethyl)-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indole-3-carboxylateas a yellow solid.

LC-MS (ES, m/z): 280 [M+H]+. 1H-NMR-300 MHz, CDCl3, ppm): δ 5.73 (s,1H), 4.77 (s, 2H), 4.09 (s, 3H), 3.95 (s, 3H), 3.85 (s, 3H).

Example 24E: Synthesis of Quinone 5

Quinone 5 was prepared according to the following reaction scheme:

Into a 250-mL round-bottom flask was placed a solution of5-methoxy-1H-indole-2-carboxylic acid (10 g, 52.31 mmol, 1.00 equiv) inmethanol (100 mL), followed by the addition of thionyl chloride (12.5 g,105.07 mmol, 2.00 equiv) dropwise with stirring. The resulting solutionwas heated to reflux for 4 h, cooled to room temperature andconcentrated under vacuum to afford 11 g (crude) of methyl5-methoxy-1H-indole-2-carboxylate as a gray solid.

Into a 5-L 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed N,N-dimethylformamide (2 L),followed by the addition of sodium hydride (37.6 g, 1.10 mol, 1.50equiv, 70%) in several batches with stirring. To this was added methyl5-methoxy-1H-indole-2-carboxylate (150 g, 730.96 mmol, 1.00 equiv)dropwise with stirring at less than 10° C. The mixture was stirred for0.5 h. To the mixture was added MeI (125 g, 0.88 mol, 1.20 equiv)dropwise with stirring. The resulting solution was stirred overnight atroom temperature and diluted with 5 L of water. The solids werecollected by filtration, washed with 3×1 L of water and dried to afford163 g (crude) of methyl 5-methoxy-1-methyl-1H-indole-2-carboxylate as ayellow solid.

Into a 5000-mL 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of LiAlH₄ (111 g,2.92 mol, 4.00 equiv) in tetrahydrofuran (1500 mL), followed by theaddition of a solution of methyl5-methoxy-1-methyl-1H-indole-2-carboxylate (160 g, 729.81 mmol, 1.00equiv) in tetrahydrofuran (1000 mL) dropwise with stirring at 0° C. over30 min. The mixture was stirred at 0° C. for 1 h and at room temperaturefor 3 h. The mixture was then quenched by the addition of 111 g ofwater, 333 mL of aqueous NaOH (15%) and 111 g of water at 0° C. Thesolids were filtered out. The filtrate was dried over anhydrous sodiumsulfate and concentrated under vacuum to afford 100 g (72%) of(5-methoxy-1-methyl-1H-indol-2-yl)methanol as a yellow solid.

Into a 3000-mL 4-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed a solution of(5-methoxy-1-methyl-1H-indol-2-yl)methanol (100 g, 522.94 mmol, 1.00equiv) in dichloromethane (2000 mL), followed by the addition oftriethylamine (61.6 g, 608.76 mmol, 1.50 equiv) dropwise with stirringat room temperature. The mixture was stirred for 30 min. To this wasadded acetyl chloride (79 g, 1.01 mol, 1.50 equiv) dropwise withstirring at room temperature. The resulting solution was stirred at roomtemperature for 3 h and concentrated under vacuum. The residue waspurified on a silica gel column eluting with ethyl acetate: petroleumether (1:10-1:5) to afford 75 g (61%) of(5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate as a yellow solid.

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed N,N-dimethylformamide (20 g,273.64 mmol, 6.00 equiv), followed by the addition of POCl₃ (9.85 g,0.0642 mol, 1.50 equiv) dropwise with stirring at 0° C. The mixture wasstirred at room temperature for 30 min. To this was added(5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate (10 g, 42.87 mmol, 1.00equiv) in portions with stirring at less than 0° C. The resultingsolution was stirred at room temperature for 2 h and quenched by theaddition of 100 mL of water/ice. The pH value of the solution wasadjusted to 7-8 with aqueous sodium hydroxide (2 N). The resultingsolution was extracted with 3×200 mL of ethyl acetate. The organiclayers were combined, dried over anhydrous sodium sulfate andconcentrated under vacuum. The residue was purified on a silica gelcolumn eluting with ethyl acetate: petroleum ether (1:3) to afford 9 g(80%) of (3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate as ayellow solid.

Into a 250-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed(3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate (9 g, 3.60mmol, 1.00 equiv), AcOH (100 mL), followed by the addition of a solutionof HNO3 (20 mL) in AcOH (50 mL) dropwise with stirring at less than 5°C. The resulting solution was stirred at room temperature for 30 min,diluted with 1000 mL of water and stirred for 30 min. The solids werecollected by filtration, washed with 3×100 mL of water and dried toafford 8.6 g (crude) of(3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl)methyl acetate as alight red solid.

Into a 1000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed(3-formyl-5-methoxy-1-methyl-4-nitro-1H-indol-2-yl)methyl acetate (8 g,26.12 mmol, 1.00 equiv), ethanol (400 mL), followed by the addition ofSn (34.1 g, 11.00 equiv) in portions with stirring at 0° C. To this wasadded hydrogen chloride (4 N) (400 mL) dropwise with stirring. Theresulting solution was stirred at 0° C. for 2 h, concentrated undervacuum and diluted with 500 mL of water. The pH value of the solutionwas adjusted to 7-8 with saturated aqueous sodium bicarbonate. Thesolids were filtered out and washed with 3×50 mL of EA. The filtrate wasextracted with 4×200 mL of ethyl acetate. The organic layers werecombined, dried over anhydrous sodium sulfate and concentrated undervacuum. The residue was purified on a silica gel column eluting withethyl acetate: petroleum ether (1:2) to afford 6.5 g (90%) of(4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate as ayellow solid.

Into a 2000-mL 3-necked round-bottom flask purged and maintained with aninert atmosphere of nitrogen was placed(4-amino-3-formyl-5-methoxy-1-methyl-1H-indol-2-yl)methyl acetate (6 g,21.72 mmol, 1.00 equiv), acetone (600 mL), followed by the addition of asolution of (KO₃S)₂NO (17.48 g, 65.2 mol, 3.00 equiv) in NaH₂PO₄ (0.4 M)(1200 mL) dropwise with stirring at less than 10° C. The resultingsolution was stirred at room temperature for 2 h and concentrated undervacuum. The residue was extracted with 3×300 mL of dichloromethane. Theorganic layers were combined, washed with 3×300 mL of water, dried overanhydrous sodium sulfate and concentrated under vacuum. The residue waspurified on a silica gel column eluting with ethyl acetate: petroleumether (1:2) to afford 3.5 g (55%) of(3-formyl-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indol-2-yl)methylacetate as a yellow solid.

Into a 250-mL 3-necked round-bottom flask was placed(3-formyl-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indol-2-yl)methylacetate (3.5 g, 12.02 mmol, 1.00 equiv), dichloromethane (47 mL),followed by the addition of a solution of LiOH (380 mg, 15.87 mmol, 1.30equiv) in methanol (40 mL) dropwise with stirring. The resultingsolution was stirred at room temperature for 30 min, diluted with 80 mLof DCM and washed with 3×100 mL of water. The organic phase was driedover anhydrous sodium sulfate and concentrated under vacuum. The residuewas purified on a silica gel column eluting with ethyl acetate:petroleum ether (1:2) to afford 1.1 g (37%) of2-(hydroxymethyl)-5-methoxy-1-methyl-4,7-dioxo-4,7-dihydro-1H-indole-3-carbaldehydeas a yellow solid.

LC-MS (ES, m/z): 250 [M+H]+. 1H-NMR (CDCl3, 300 MHz, ppm): □ 10.55 (s,1H), 5.79 (s, 1H), 4.83 (s, 2H), 4.09 (s, 3H), 3.89 (s, 3H).

Example 24F: Synthesis of Quinone 6

Quinone 6 was prepared according to the following reaction scheme:

To a stirred solution of compound 1 (30 mg, 0.135 mmol) in dry DMF (2.0mL) was added bis(4-nitrophenyl) carbonate (82.5 mg, 0.271 mmol) andDIEA (87.5 mg, 0.678 mmol) at 0° C., then the mixture was stirred at 25°C. for 2 h under N₂. The mixture was used for next step without furtherpurification.

To a mixture of above was added compound 3 (100.2 mg, 0.814 mmol), DIEA(87.6 mg, 0.678 mmol), DMF (2.0 mL) and catalytic amount of HOBt (5.0mg) at 0° C. Then the mixture was stirred at 25° C. for 15 h under N₂.It was diluted with water (15 mL), and extracted it with EtOAc (30mL×4). The combined organic phase was washed it with brine (30 mL),dried over Na₂SO₄, and concentrated to give the desired product (26 mg,52%) as a red solid. LCMS: (5-95 AB, 1.5 min), 0.714 min, MS=392.8[M+23].

To a solution of compound 4 (26 mg, 0.070 mmol) in dry DMF (1.0 mL) wasadded bis(4-nitrophenyl) carbonate (42.7 mg, 0.140 mmol) and DIEA (45.4mg, 0.351 mmol) at 0° C. Then the mixture was stirred at 25° C. for 15 hunder N₂. The mixture was used for next step without furtherpurification.

To the solution of compound 5 (37.59 mg, 0.070 mmol) in dry DMF (1.0 mL)was added norfloxacin (44.83 mg, 0.140 mmol) and DIEA (45.36 mg, 0.351mmol) at 0° C. The mixture was stirred at 25° C. for 2 h under N₂. Itwas filtered and the filtrate was purified by prep-HPLC (FA) to give theproduct (30 mg, 59.7%) as a yellow solid. LCMS: (5-95, AB, 1.5 min),RT=0.862 min, MS=715.9[M+1]+; 1H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H),8.95 (s, 1H), 8.28 (s, 1H), 7.95-7.93 (d, J=8.0 Hz, 1H), 7.46-7.44 (d,J=8.0 Hz, 2H), 7.33-7.31 (d, J=8.0, 2H), 7.19 (s, 1H), 6.68 (s, 1H),5.85 (s, 1H), 5.22 (s, 2H), 5.04 (s, 2H), 4.57 (s, 2H), 3.96 (s, 3H),3.77 (s, 3H), 3.60 (s, 8H), 1.40 (s, 3H).

Example 24G: Synthesis of Quinone 7

Quinone 7 was prepared according to the following reaction scheme:

To a solution of compound 1 (100 mg, 0.45 mmol) in dry DMF (10 mL) wasadded bis(4-nitrophenyl) carbonate (280 mg, 0.9 mmol) and DIEA (175 mg,1.36 mmol) at 30° C. The mixture was stirred at 30° C. for 16 h underN₂. It was used in the next step without further purification.

To a solution of compound 2, norfloxacin (288 mg, 0.9 mmol) in dry DMF(10.0 mL) was added DIEA (116 mg, 0.90 mmol) at 30° C. After the mixturewas stirred at 30° C. for 2 h, it was filtered and the filter cake waswashed with DCM/MeOH (10/1) and then concentrated to give the desiredproduct as a yellow solid (150 mg, 59%).

LCMS: (5-95, AB, 1.5 min), RT=0.824 min, MS=566.9[M+1]; 1H NMR (400 MHz,DMSO-d6) δ 8.89 (s, 1H), 7.94 (d, J=13.2 Hz, 1H), 7.20 (d, J=7.2 Hz,1H), 6.63 (s, 1H), 5.83 (s, 1H), 5.22 (s, 2H), 4.57 (d, J=6.8 Hz, 2H),3.97 (s, 2H), 3.80 (s, 2H), 3.63 (s, 3H), 3.37 (s, 3H), 3.21 (s, 4H),1.46 (d, J=7.2 Hz, 3H).

Example 24H: Synthesis of Quinone 8

Quinone 8 was prepared according to the following reaction scheme:

The alcohol (100 mg, 0.4 mmol) was dissolved in DMF (2 mL).p-nitrophenylcarbonate (610 mg, 2.0 mmol, 5 eq.) was added, followed byDIPEA (0.35 mL, 2.0 mmol, 5 eq.) and the reaction was stirred for 3 h atrt. The reaction was concentrated and carried on crude.

Norfloxacin (130 mg, 0.4 mmol, 1 eq.) was added to a vial, followed bythe starting carbonate (200 mg, 0.4 mmol), followed by DMF (3 mL). HOBt(10 mg, 0.08 mmol, 0.2 eq.), then pyridine (0.3 mL, 4 mmol, 10 eq.)added and the reaction was stirred at rt for 19 h. The reaction waspurified by HPLC to give 136 mg of the product (57% over two steps).

1H NMR (400 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.96 (s, 1H), 7.94 (d, J=13.2Hz, 1H), 7.20 (d, J=7.2 Hz, 1H), 5.99 (s, 1H), 5.49 (s, 2H), 4.58 (q,J=7.1 Hz, 2H), 4.02 (s, 3H), 3.83 (s, 3H), 3.63-3.48 (m, 5H), 2.07 (s,3H), 1.41 (t, J=7.1 Hz, 3H).

Example 24I: Synthesis of Quinone 9

Quinone 9 was prepared according to the following reaction scheme:

The alcohol (200 mg, 0.9 mmol) was dissolved in DMF (4 mL).p-nitrophenylcarbonate (284 mg, 1 mmol, 1.1 eq.) was added, followed byDIPEA (0.3 mL, 1.7 mmol, 2 eq.) and the reaction was stirred for 19 h atrt. The reaction was concentrated and carried on crude.

Norfloxacin (290 mg, 0.9 mmol, 1 eq.) was added to a vial, followed bythe starting carbonate (350 mg, 0.9 mmol), followed by DMF (4 mL). HOBt(25 mg, 0.18 mmol, 0.2 eq.), then pyridine (0.74 mL, 9 mmol, 10 eq.)added and the reaction was stirred at rt for 24 h. The reaction waspurified by HPLC to give 103 mg of the product (20% over two steps).

1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H), 7.98 (d, J=12.8 Hz, 1H), 7.31(s, 1H), 7.24 (d, J=7.0 Hz, 1H), 5.86 (s, 1H), 5.19 (s, 2H), 4.61 (d,J=9.9 Hz, 4H), 3.92 (s, 3H), 3.80 (s, 3H), 3.63 (s, 6H), 1.58-1.33 (m,3H).

Example 24J: Synthesis of Quinone 10

Quinone 10 was prepared according to the following reaction scheme:

The alcohol (100 mg, 0.43 mmol) was dissolved in DMF (2 mL).p-nitrophenylcarbonate (142 mg, 0.47 mmol, 1.1 eq.) was added, followedby DIPEA (0.15 mL, 0.85 mmol, 2 eq.) and the reaction was stirred for 19h at rt. The reaction was concentrated and carried on crude.

Norfloxacin (136 mg, 0.43 mmol, 1 eq.) was added to a vial, followed bythe starting carbonate (170 mg, 0.43 mmol), followed by DMF (7 mL). HOBt(12 mg, 0.09 mmol, 0.2 eq.), then pyridine (0.35 mL, 4.3 mmol, 10 eq.)added and the reaction was stirred at rt for 24 h. The reaction waspurified by HPLC to give 18 mg of the product (7% over two steps).

1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.48 (s, 1H), 7.94 (d, J=13.0Hz, 1H), 7.20 (d, J=7.1 Hz, 1H), 6.80 (s, 1H), 5.78 (s, 1H), 5.17 (s,2H), 4.58 (d, J=7.7 Hz, 2H), 3.86 (s, 3H), 3.76 (s, 4H), 3.53 (s, 5H),2.28 (s, 3H), 1.40 (t, J=7.0 Hz, 3H).

Example 24K: Synthesis of Quinone 11

Quinone 11 was prepared according to the following reaction scheme:

The alcohol (100 mg, 0.43 mmol) was dissolved in DMF (2 mL).p-nitrophenylcarbonate (142 mg, 0.47 mmol, 1.1 eq.) was added, followedby DIPEA (0.15 mL, 0.85 mmol, 2 eq.) and the reaction was stirred for 19h at rt. The reaction was concentrated and carried on crude.

Norfloxacin (136 mg, 0.43 mmol, 1 eq.) was added to a vial, followed bythe starting carbonate (170 mg, 0.43 mmol), followed by DMF (7 mL). HOBt(12 mg, 0.09 mmol, 0.2 eq.), then pyridine (0.35 mL, 4.3 mmol, 10 eq.)added and the reaction was stirred at rt for 24 h. The reaction waspurified by HPLC to give 31 mg of the product (12% over two steps).

1H NMR (400 MHz, DMSO-d6) δ 8.95 (s, 1H), 8.49 (s, 2H), 7.94 (d, J=13.1Hz, 1H), 6.76 (s, 2H), 5.19 (s, 2H), 4.58 (d, J=7.5 Hz, 2H), 3.96 (s,3H), 3.83-3.69 (m, 5H), 3.58 (t, J=5.1 Hz, 4H), 2.30 (s, 3H), 1.40 (t,J=7.1 Hz, 3H).

Example 24L: Synthesis of Quinone 12

Quinone 12 was prepared according to the following reaction scheme:

The alcohol (100 mg, 0.36 mmol) was dissolved in DMF (2 mL).p-nitrophenylcarbonate (545 mg, 1.8 mmol, 5 eq.) was added, followed byDIPEA (0.31 mL, 1.8 mmol, 5 eq.) and the reaction was stirred for 3 h atrt. The reaction was concentrated and carried on crude.

Norfloxacin (110 mg, 0.36 mmol, 1 eq.) was added to a vial, followed bythe starting carbonate (160 mg, 0.36 mmol), followed by DMF (3 mL). HOBt(10 mg, 0.07 mmol, 0.2 eq.), then pyridine (0.29 mL, 3.6 mmol, 10 eq.)added and the reaction was stirred at rt for 19 h. Add more norfloxacin(110 mg, 0.36 mmol, 1 eq.) and the reaction was stirred for 2.5 days.The reaction was purified by HPLC to give 89 mg of the product (40% overtwo steps).

1H NMR (400 MHz, DMSO-d6) δ 8.96 (s, 1H), 7.94 (d, J=13.1 Hz, 1H), 7.20(d, J=7.2 Hz, 1H), 5.93 (s, 1H), 5.32 (s, 2H), 4.58 (q, J=7.1 Hz, 2H),4.00 (s, 3H), 3.80 (d, J=3.3 Hz, 6H), 3.64-3.49 (m, 8H), 1.41 (t, J=7.1Hz, 3H).

Example 25: Synthesis of PBD Dimer Diaphorase Prodrug 1 Comprising aLinker for Conjugation to an Antibody

PBD dimer diaphorase prodrug 1 comprising a linker was preparedaccording to the following reaction scheme:

Each asterisk in the above structure, and elsewhere depicted in Example25, represents a chiral center.

To a solution of compound A1 (1.00 g, 1.17 mmol) in DCM (30 mL) wasadded a solution of triphosgene (347 mg, 1.17 mmol) and Et₃N (356 mg,3.52 mmol) in DCM (10 mL) at 30° C. After the mixture was stirred at 30°C. for 30 min, it was concentrated, and dibutyltin diacetate (0.32 mL,1.22 mmol) was added, followed by a solution of Compound A2 (215 mg,0.97 mmol) and Et₃N (369 mg, 3.65 mmol) in DMF (15 mL). The mixture wasstirred at 25° C. for 1 h. The mixture was diluted with water (10 mL)and stirred for 20 min. Then the mixture was concentrated and purifiedby flash column chromatography (0-5% MeOH in DCM) to give Compound A3(800 mg, 26.9%) as a yellow solid. LCMS (5-95, AB, 1.5 min): RT=1.032min, m/z=1100.5 [M+1]+.

To a solution of triphosgene (97.1 mg, 0.33 mmol) in DCM (15 mL) wasadded a solution of Compound A3 (800.0 mg, 0.33 mmol) and Et3N (99.31mg, 0.98 mmol) in DCM (5.0 mL). After the mixture was stirred at 30° C.for 30 min, it was concentrated. To above residue was added a solutionof MC_VC_PAB (368.0 mg, 0.33 mmol) and Et₃N (0.14 mL, 0.98 mmol) in DMF(15 mL). The mixture was stirred at 25° C. for 12 h. The mixture wasconcentrated and purified by flash column chromatography (5-10% MeOH inDCM) to give A4 (300 mg, 37%) as a yellow solid. LCMS (5-95, AB, 1.5min): RT=1.023 min, m/z=850.5 [M/2+1]+.

To a solution of Compound A4 (300.0 mg, 0.18 mmol) in THF (1.0 mL) wasadded water (1.0 mL) and HOAc (1.5 mL), and stirred at 25° C. for 12 h.The mixture was diluted with EtOAc (80 mL), washed with sat.NaHCO₃ (3×40mL), and concentrated to give Compound A5 (160 mg, 61.7%) as a redsolid.

To a solution of Compound A5 (140.0 mg, 0.10 mmol) in DMSO (5.0 mL) wasadded IBX (266 mg, 0.95 mmol). After the mixture was stirred at 38° C.for 12 h, it was purified by prep-HPLC (ACN 37-67%/0.225% FA in water),followed by prep-TLC (10% MeOH in DCM, Rf=0.5) to give PBD dimerdiaphorase prodrug 1 comprising a linker (15 mg, 10.5%) as a yellowsolid. LCMS (5-95, AB, 1.5 min): RT=0.712 min, m/z=734.2 [M/2+1]+.

Example 26: Synthesis of PBD Dimer Prodrug Antibody-Drug Conjugates(ADC)

ADC 1A and 1B were prepared by conjugation of an antibody and the PBDdimer ADC disulfide prodrug linker-drug intermediate of Example 21D, andhave the structure:

ADC 2A and 2B were prepared by conjugation of an antibody and the PBDdimer ADC disulfide prodrug linker-drug intermediate of Example 21B, andhave the structure:

ADC 3 were prepared by conjugation of an antibody and the PBD dimer ADCdisulfide prodrug linker-drug intermediate of Example 21A, and have thestructure:

ADC 4 were prepared by conjugation of an antibody and the PBD dimer ADCdisulfide prodrug linker-drug intermediate of Example 21C, and have thestructure:

ADC 5 were prepared by conjugation of an antibody and the PBD dimer ADCdisulfide prodrug linker-drug intermediate of Example 21E, and have thestructure:

PBD Dimer ADC boronic acid prodrug 1A and 1B are prepared by conjugationof an antibody and the PBD dimer ADC boronic acid prodrug linker-drugintermediate of Example 22A, and have the structure:

PBD Dimer ADC diaphorase prodrug 1A and 1B are prepared by conjugationof an antibody and the PBD dimer ADC diaphorase prodrug linker-drugintermediate of Example 25, and have the structure:

When introducing elements of the present disclosure or the preferredembodiments(s) thereof, the articles “a”, “an”, “the” and “said” areintended to mean that there are one or more of the elements. The terms“comprising”, “including” and “having” are intended to be inclusive andmean that there may be additional elements other than the listedelements.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims.

The invention claimed is:
 1. A pyrrolobenzodiazepine prodrugdimer-antibody conjugate compound of formula (I) comprising a firstpyrrolobenzodiazepine prodrug monomer M1 and a secondpyrrolobenzodiazepine-antibody monomer M2

wherein: (I) for M1 (a) R² is selected from —H, ═CH₂, —CN, —R, ═CH—R,aryl, heteroaryl, bicyclic ring and heterobicyclic ring; (b) R³ is H;(c) R⁶, R⁷ and R⁹ are independently selected from H, R, OH, OR, halo,amino, nitro, SH and SR; (d) X is selected from S, O and NH; (e) R¹⁰ isa prodrug moiety comprising (i) a glutathione-activated disulfide, or(ii) a reactive oxygen species-activated aryl boronic acid or arylboronic ester; R¹¹ is selected from (i) H and R when X is O or NH, and(ii) H, R and O_(z)U when X is S, wherein z is 2 or 3 and U is amonovalent pharmaceutically acceptable cation; (g) R is selected from alower alkyl group having 1 to 10 carbon atoms and an arylalkyl group ofup to 12 carbon atoms, (i) wherein the alkyl group optionally containsone or more carbon-carbon double or triple bonds, or an aryl group, ofup to 12 carbon atoms and (ii) wherein R is optionally substituted byone or more halo, hydroxy, amino, or nitro groups, and optionallycontains one or more hetero atoms; and (h) the dashed lines represent anoptional double bond between one of: (i) C₁ and C₂; (ii) C₂ and C₃; and(iii) C₂ and R², (II) for M2 (a) R^(2′), R^(3′), R^(6′), R^(7′), R^(9′),R^(11′) and X′ correspond to R², R³, R⁶, R⁷, R⁹, R¹¹ and X,respectively; (b) L is a self-immolative linker comprising at least oneof a disulfide moiety, a peptide moiety and a peptidomimetic moiety; and(c) the dashed lines represent an optional double bond between one of:(i) C_(1′) and C_(2′); (ii) C_(2′) and C_(3′); and (iii) C_(2′) andR^(2′), (III) M1 and M2 are bound at the C8 position by a moiety-Q-T-Q′- wherein Q and Q′ are independently selected from O, NH and S,and wherein T is an optionally substituted C₁₋₁₂ alkylene group that isfurther optionally interrupted by one or more heteroatoms and/oraromatic rings, (IV) p is 1, 2, 3, 4, 5, 6, 7 or 8, (V) Ab is anantibody, and (VI) each asterisk independently represents a chiralcenter.
 2. The compound of claim 1 wherein the bond between C₁ and C₂ ofM1 is a single bond; the bond between C₂ and C₃ of M1 is a single bond;the bond between C_(1′) and C_(2′) of M2 is a single bond; the bondbetween C_(2′) and C_(3′) of M2 is a single bond; C₃ of M1 issubstituted with two R³ groups, each of which is H; C_(3′) of M2 issubstituted with two R^(3′) groups, each of which is H; the bond betweenC₂ and R² of M1 is a double bond; and the bond between C_(2′) and R^(2′)of M2 is a double bond.
 3. The compound of claim 1 wherein R² and R^(2′)are ═CH₂.
 4. The compound of claim 1 wherein R⁶, R^(6′), R⁹ and R^(9′)are H.
 5. The compound of claim 1 wherein R⁷ and R^(7′) are OCH₃.
 6. Thecompound of claim 1 wherein Q and Q′ are O and wherein T is C₃ alkyleneor C₅ alkylene.
 7. The compound of claim 1 wherein p is 1, 2, 3, or 4.8. The compound of claim 1 comprising a mixture of conjugate compounds,wherein the average drug loading per antibody in the mixture ofconjugate compounds is about 2 to about
 5. 9. The compound of claim 8wherein the antibody comprises at least one cysteine sulfhydryl moietyengineered for conjugation, wherein the antibody binds to one or moretumor-associated antigens or cell-surface receptors selected from: (1)BMPR1B; (2) E16; (3) STEAP1; (4) MUC16; (5) MPF; (6) Napi2b; (7) Sema5b; (8) PSCA hlg; (9) ETBR; (10) MSG783; (11) STEAP2; (12) TrpM4; (13)CRIPTO; (14) CD21; (15) CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19)MDP; (20) IL20Rα; (21) Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA;(25) GEDA; (26) BAFF-R; (27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB;(31) P2X5; (32) CD72; (33) LY64; (34) FcRH1; (35) FcRH5; (36) TENB2;(37) PMEL17; (38) TMEFF1; (39) GDNF-Ra1; (40) Ly6E; (41) TMEM46; (42)Ly6G6D; (43) LGR5; (44) RET; (45) LY6K; (46) GPR19; (47) GPR54; (48)ASPHD1; (49) Tyrosinase; (50) TMEM118; (51) GPR172A; (52) CD33; and (53)CLL-1.
 10. The compound of claim 1 wherein the antibody is acysteine-engineered antibody.
 11. The compound of claim 10 wherein thecysteine-engineered antibody comprises LC K149C, HC A118C, HC A140C orLC V205C as the site of linker conjugation.
 12. The compound of claim 11wherein the antibody is selected from anti-HER2, anti-CD22, anti-CD33,anti-Napi2b, anti-Ly6E, and anti-CLL-1.
 13. The compound of claim 1wherein the linker comprises a disulfide moiety or a peptide moiety. 14.The compound of claim 1 wherein L-Ab is of the structure:

wherein (I) Ab is an antibody; (II) S_(C) is an antibody cysteine sulfuratom; (III) R⁷⁰ and R⁷¹ are independently selected from H and C₁₋₃alkyl, wherein only one of R⁷⁰ and R⁷¹ can be H, or R⁷⁰ and R⁷¹ togetherwith the carbon atom to which they are bound form a four- tosix-membered ring optionally comprising an oxygen heteroatom; (IV) thewavy line indicates the point of attachment to the oxygen atom of thecarbamate moiety of M2; and (V) p is as defined in claim
 1. 15. Thecompound of claim 14 wherein R⁷⁰ and R⁷¹ are independently selected fromH, —CH₃ and —CH₂CH₃, wherein only one of R⁷⁰ and R⁷¹ can be H, or R⁷⁰and R⁷¹ together with the carbon atom to which they are bound form aring selected from cyclobutyl, cyclopentyl, cyclohexyl, tetrahydrofuranand tetrahydropyran.
 16. The compound of claim 1 wherein R¹⁰ is an arylboronic acid or an aryl boronic ester of formula (IVa):

(I) wherein R²⁰ and R²¹ are independently selected from H, optionallysubstituted alkyl or heteroalkyl, optionally substituted cycloalkyl orheterocycloalkyl, and optionally substituted aryl or heteroaryl, or (II)wherein R²⁰ and R²¹ together are an optionally substituted moiety—(CH₂)_(n)— wherein n is 2 or 3, said moiety together with the O atomsto which they are bound and the B atom form a hetercyclooalkyl ring,wherein said heterocycloalkyl ring may optionally comprise a fusedheteroalkyl ring, a fused aryl ring or a fused heteroaryl ring, and(III) wherein the wavy line indicates the point of attachment to theoxygen atom of the carbamate moiety of M1 of formula (I).
 17. Thecompound of claim 16 wherein R¹⁰ is selected from formulae (IVb) and(IVc):

(I) wherein R³⁰, R³¹, R³², R³³, R⁴⁰, R⁴¹, R⁴², R⁴³, R⁴⁴ and R⁴⁵ areindependently selected from H, halogen, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₂Cl, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂,optionally substituted C₁₋₈ alkyl or heteroalkyl, optionally substitutedcycloalkyl or heterocycloalkyl comprising from 2 to 7 carbon atoms,optionally substituted aryl or heteroaryl, or (II) wherein (i) one ofR³⁰ or R³¹ and one of R³² or R³³, (ii) one of R⁴⁰ or R⁴¹ and one of R⁴²or R⁴³, and/or (iii) one of R⁴² or R⁴³ and one of R⁴⁴ or R⁴⁵ form anoptionally substituted fused cycloalkyl ring, fused heterocycloalkylring, fused aryl ring or fused heteroaryl ring having from 2 to 7 carbonatoms.
 18. The compound of claim 16 wherein R¹⁰ is selected fromformulae (IVd) to (IVi):


19. The compound of claim 18 wherein R¹⁰ is formula (IVd).
 20. Thecompound of claim 1 wherein R¹⁰ is a disulfide of formula (V):

(I) wherein R⁵⁰ is optionally substituted C₁₋₈ alkyl or heteroalkyl andR⁵¹ is optionally substituted C₂ alkylene or optionally substitutedbenzylene; and (II) wherein the wavy line indicates the point ofattachment to the oxygen atom of the carbamate moiety of M1 of formula(I).
 21. The compound of claim 20 wherein R⁵⁰ is C₂₋₆ alkyl, optionallysubstituted with OH, and R⁵¹ is of the formula (Va):

(I) wherein R⁶¹ and R⁶² are independently selected from H and CH₃, orR⁶¹ and R⁶² together with the carbon atom to which they are bound forman optionally substituted cycloalkyl, optionally substitutedheterocycloalkyl, optionally substituted aryl or optionally substitutedheteroaryl comprising from 2 to 6 carbon atoms; (II) wherein R⁶³ and R⁶⁴are independently selected from H and CH₃; and (III) wherein the wavyline at the carbon atom bearing R⁶³ and R⁶⁴ indicates the point ofattachment to the oxygen atom of the carbamate moiety of M1 of formula(I).
 22. The compound of claim 21 wherein R⁶³ and R⁶⁴ are H.
 23. Thecompound of claim 20 wherein: (I) R⁵⁰ is selected from CH₃CH₂—,HOCH₂CH₂—, (CH₃)₂CH— and (CH₃)₃C—; and (II) R⁵¹ is selected fromformulae (Vb) to (Vf):


24. The compound of claim 1 of formula (VIa):


25. The compound of claim 1 selected from:

wherein p is 1, 2, 3, or
 4. 26. The compound of claim 25 wherein Ab isan antibody that binds to one or more tumor-associated antigens orcell-surface receptors selected from: (1) BMPR1B; (2) E16; (3) STEAP1;(4) MUC16; (5) MPF; (6) Napi2b; (7) Sema 5b; (8) PSCA hlg; (9) ETBR;(10) MSG783; (11) STEAP2; (12) TrpM4; (13) CRIPTO; (14) CD21; (15)CD79b; (16) FcRH2; (17) HER2; (18) NCA; (19) MDP; (20) IL20Rα; (21)Brevican; (22) EphB2R; (23) ASLG659; (24) PSCA; (25) GEDA; (26) BAFF-R;(27) CD22; (28) CD79a; (29) CXCR5; (30) HLA-DOB; (31) P2X5; (32) CD72;(33) LY64; (34) FcRH1; (35) FcRH5; (36) TENB2; (37) PMEL17; (38) TMEFF1;(39) GDNF-Ra1; (40) Ly6E; (41) TMEM46; (42) Ly6G6D; (43) LGR5; (44) RET;(45) LY6K; (46) GPR19; (47) GPR54; (48) ASPHD1; (49) Tyrosinase; (50)TMEM118; (51) GPR172A; (52) CD33; and (53) CLL-1.
 27. A pharmaceuticalcomposition comprising the pyrrolobenzodiazepine prodrug dimer-antibodyconjugate compound of according to claim 1 and a pharmaceuticallyacceptable diluent or excipient.
 28. A method of treating cancercomprising administering to a patient in need of treatment thepharmaceutical composition of claim
 27. 29. An article of manufacturecomprising a pharmaceutical composition of claim 27, a container, and apackage insert or label indicating that the pharmaceutical compositioncan be used to treat cancer.